Abstract:[Objectives] Four species of amphibian were recorded in Wanfoshan Town, Tongdao County, Hunan Province, China at 26°11′47″ N, 109°56′21″ E, altitude 924 m during a filed survey on 23 May, 2020. They are recognized as first recorded in Hunan province by morphological and molecular. [Methods] [Objectives] The genus Nidirana species was endemic group of classes in Oriental realm, widely distributed in subtropical areas of East and Southeast Asia. Among the species in genus Nidirana, Nidirana adenopleura had the widest distribution area and only distributed in Taiwan, northern of Fujian, southern of Zhejiang, central Jiangxi and other regions in China. The recent contribution to the phylogeny of genus Nidirana reconsidered the previously recorded as N. adenopleura populations in southern of China were actually the genus Nidirana new species. There are several geographical population of genus Nidirana mainly distributed in the northeast, north, central and southern regions of Guangxi, had been certified the genus Nidirana population form Dupang mountain was N. xiangica, the genus Nidirana population form Dayao mountain was N. yaoica, and the genus Nidirana population form Daming mountain was N. guangxiensis. [Methods] Seven male and three female specimens collected form Rongshui were examined and measured with digital calipers. The measurements were as follows: snout-vent length, head length, head width, snout length, internasal distance, interorbital distance, eye diameter, tympanum diameter, tympanum-eye distance, hand length, radio-ulna length, foot length, tibial length, and compared characters with the N. leishanensis specimens obtained from Guizhou. All specimens were fixed in 10% buffered formalin, transferred to 70% ethanol, and de?posited in NHMG. Four muscular samples attained from euthanasia specimens and then preserved in 95% ethanol and stored at ﹣40 ℃ were used for molecular analysis. All samples were 36 sequences from all known Nidirana species and two sequences from the out-group Babina were obtained from GenBank and incorporated into our dataset (Detail information of these materials was shown in Table 1). Genomic DNA were extracted from muscle tissue samples using DNA extraction kit. A mitochondrion genes namely partial cytochrome C oxidase I gene (COI) were am?plified, and the primers were dgLCO and dgHCO. PCR amplifications were processed with the cycling conditions that initial denaturing step at 95 ℃ for 4 min, 35 cycles of denaturing at 94 ℃ for 40 s, annealing at 52 ℃ for 40 s and extending at 72 ℃ for 60 s, and a final extending step at 72 ℃ for 10 min. PCR products were sequenced with both forward using reverse primers. The Clustal W algorithm was used in MEGA 6.0 software to compare all the COI gene sequences and calculate the genetic distance between specimens and other species by the p-distance model. The comparison sequences were calculated to get the optimal nucleotide substitution model in jmodeltest v2.1.2. Sequenced data analyzed using maximum likelihood (ML) in RaxmlGUI 1.3 to structure the phylogenetic tree. Advertisement calls of the specimens were recorded in the field at the air temperature 22 ℃ using a SONY PCM D100 digital sound recorder. Praat 6.0.27 was used to obtain the oscillogram, sonogram, and power spectrum. Raven pro 1.5 was used to quantify the acoustic properties. [Results] We obtained four mitochondrial COI gene sequences length 639 bp and the optimal nucleotide substitution model for phylogenetic analysis was GTR + G + I. The Maximum likelihood phylogenetic tree of genus Nidirana showed these samples and N. leishanensis samples in Guizhou together formed a monophyletic group (Fig. 1). Genetic distance estimation based on p-distance model showed the intraspecific genetic distance was 0.2%, and the genetic distance was 0.5% between the specimens and the N. leishanensis samples (Table 2). The following principal characters of genus Nidirana specimens: a largebody size (Snout-vent length, SVL, males: 47.5 mm < SVL < 51.2 mm; females: 49.4 mm < SVL < 59.1 mm, for more please see Table 3); the presence of lateroventral grooves both on fingers and toes, relative finger lengths: II < IV < I < III; tibiotarsal articulation reaching the level between eye and nostril when leg stretched forward; a pair of subgular internal vocal sacs at corners of throat in males; nuptial pad present on the inner side of base of fingers I and II in males in breading season; webbing formula: I 2 –213 II 2 –223 III 312 –323 IV 323 –3V. Dorsal skin of head, anterior part of body, ventral surface of head and limbs were smooth. All specimens were similar in morphology but some individuals different in color pattern (Fig. 2). The advertisement call had only one strophe with one syllable. The time of syllable duration and interval syllables duration were respectively 0.18﹣0.25 s, 11.40﹣23.08 s, and the frequency of sound wave was 350﹣4 270 Hz (Fig. 3). With this wok, the morphological characteristics and acoustic characteristics of genus Nidirana population form Rongshui were accorded with N. leishanensis. We confirmed these were N. leishanensis, and it was a new record of amphibians in Guangxi. [Conclusion] This discovery extended the distribution area of the N. leishanensis from eastern Guizhou to northern Guangxi and provided important information for the phylogeography and phylogeography of genus Nidirana. We have clarified up the current species distribution of genus Nidirana in Guangxi. The complex rivers and mountain systems in Guangxi may have played an important role in isolating the communication of genus Nidirana population, promoted the population differentiation and formed new species.