During the survey conducted in Zhushadong Village (38°10′48″ N, 113°55′22″ E, 509 m), Jingxing County, Shijiazhuang City, Hebei Province, China in July 2023, a collective count of sixteen Horseshoe Bat (Rhinolophus spp.) and four Myotis (Myotis spp.) were successfully captured utilizing harp traps. The external morphological features, specifically the forearm length, of a total of twenty bats were quantified. Following this, a subset comprising four Horseshoe Bat specimens and two Myotis specimens were chosen for the purpose of measuring skull parameters, documenting echolocation calls, and conducting identification and phylogenetic analysis using COI and Cyt b gene sequences. The results of these measurements were reported as mean ± standard deviation. The echolocation calls emitted by the Horseshoe Bat were classified as frequency modulation-constant frequency-frequency modulation type (Fig. 1a). These calls exhibited a peak frequency of 54.23 ± 0.39 kHz. The forearm lengths of the bats ranged from 45.51 to 49.23 mm (Table 1). The bats possessed large ears (Fig. 2), with lengths ranging from 22 to 26.22 mm. In the Hebei samples, the third metacarpal bone was comparatively shorter, while the fourth and fifth metacarpal bones were longer. The total length of the skull varied between 18.3 and 19.26 mm (Table 1). Additionally, the bats displayed a narrow interorbital breadth. The coronoid process of the mandible was relatively low, and the second premolar of the maxilla exhibited well-developed characteristics (Fig. 2). The external morphology and skull characteristics of the Horseshoe Bat samples were found to be consistent with those of the Big-eared Horseshoe Bat (R. macrotis). Additionally, the COI gene sequences exhibited a 100% similarity with the R. macrotis in NCBI database, while the Cyt b gene sequences exhibited a 98.68% similarity, and they formed a cluster with other R. macrotis specimens in the phylogenetic tree (Fig. 4). The echolocation calls of the Myotis bats were classified as frequency modulation type (Fig. 1b), with a peak frequency of 43.56 ± 3.73 kHz. The forearm lengths ranged from 57.56 to 61.28 mm (Table 2). The ears were characterized by their narrow and elongated shape (Fig. 3), measuring between 21.19 and 23.34 mm in length. Finally, the lengths of the third, fourth, and fifth metacarpal bones were also recorded. The mandibular incisors exhibited a tricuspid morphology, with the third lower incisor in close proximity to the canine tooth. Additionally, the second lower molar displayed the smallest size (Fig. 3). The external morphology and cranial characteristics of the Myotis specimens were found to be in accordance with those of the Lesser Mouse-eared Myotis (M. blythii). Moreover, the COI gene sequence demonstrated a 99.5% similarity with the M. blythii in NCBI database, while the Cyt b gene sequence exhibited a 99.9% similarity. Furthermore, these sequences formed a cluster with the M. blythii species in the phylogenetic tree (Fig. 5).The captured species were identified as R. macrotis and M. blythii through the examination of external morphological data, skull parameters, and COI and Cyt b gene sequences. This finding signifies a novel addition to the existing knowledge of the distribution of these species within the Chiroptera of Hebei Province, China.