[Objectives] In China, Myotis horsfieldii was only to know distributed in Guangdong, Hainan and Hong Kong. This study aims to expand knowledge of geographical range of this species. [Methods] Two bat specimens (2♂: 211521 and 211540) were captured in the crack of the Datuo Station Interchange Bridge (112°57′24.4″ E, 28°3′18.1″ N, 44 m above sea level) and the Zhaohua Xiangjiang River Bridge (112°59′33.9″ E, 27°56′8.7″ N, 39 m above sea level) in August, 2021 in Changsha City, Hunan Province, respectively. The morphological and skull features of the two specimens were measured using electronic digital caliper and combined with the data of NCBI for constructed phylogenetic tree using maximum likelihood in MEGA7.0 based on Cyt b gene sequences. [Results] The main diagnosis characteristics of the two specimens as follows. Medium to small body size, forearm lengths of 36.1 mm (211521) and 33.1 mm (211540) and head body lengths of 44.0 mm and 41.2 mm, respectively. Hind-foot lengths of 10.5 mm and 10.4 mm more than half of the tibia lengths of 16.4 mm and 16.2 mm, respectively (Fig. 1, Table 2). Narrow and long skull, greatest lengths of skull 15.5 mm and 15.0 mm, breadths of braincase 7.8 mm and 7.5 mm, respectively. Delicate skull with a marked inclination at the frontal bone, and the braincase is higher than the maxilla. Zygomatic arch is thinner (Fig. 2, Table 2). Phylogenetic analysis based on Cyt b gene sequences showed that the captured bats were well clustered with M. horsfieldii formerly discovered in Hong Kong, China or Malaysia, and the genetic distance was only 0.9% to the specimen from Hong Kong, China (Fig. 3, Table 1). [Conclusion] Therefore, the two bats were confirmed to M. horsfieldii, a new record for Hunan Province. The specimens were kept in Institute of Zoology, Guangdong Academy of Science.