To survey the biodiversity of Chiroptera in northwestern China, thirteen morphologically consistent specimens were collected in August 2023, and August to September 2024 in Xinjiang, Qinghai, and Ningxia, China. The specimens’ morphological and skull characteristics were determined using electronic digital calipers. A phylogenetic tree was constructed based on the Cyt b gene sequence using the maximum likelihood method, and genetic distances were calculated using MEGA7.0 software. The main physical characteristics are as follows:head and body length 61.28 ~ 78.88 mm, forearm length 42.72 ~ 51.65 mm (Appendix 1); ear tips slightly rounded, tongue-shaped, tragus short, less than half the length of the ear; male penis gradually flattened from the base to the end, nearly triangular, and with a small groove at the end; dorsal hairs brownish-yellow as a whole, and ventral hairs grayish-white (Fig. 1). The cranium is 19.65 ~ 21.13 mm in total length (Appendix 2), and the zygomatic arches are thin, slightly widened in the middle (Fig. 2). These above characteristics are consistent with those of Botta’s Serotine (Eptesicus bottae). Phylogenetic analyses using the mitochondrial Cyt b gene molecular marker revealed that the species clustered with Botta’s Serotine from other countries (Fig. 3). The genetic distance to the named subspecies of Botta’s Serotine was only 0.4% (Table 2), which is much smaller than the distances between other species in the genus and between subspecies within the species. Combining morphological and phylogenetic analyses, these bats were identified as Botta’s Serotine. This study confirms the distribution of this species in Xinjiang, Qinghai, and Ningxia, China for the first time.