In order to elucidate the chiropteran diversity in East and North China, a series of field surveys were conducted in 2023. In March and June 2023, ten Mytios bats (1 male, 9 females) were captured by harp trap net and mist net during the field surveys in Liandu District of Lishui City, Zhejiang Province (28°29′54″ N, 119°54′35″ E, 228 m above sea level) and Shitai County of Chizhou City, Anhui Province (30°14′29″ N, 117°30′6″ E, 95 m above sea level). Morphological and phylogenetic analyses were performed to identify the species of these specimens. The specimens are medium-sized, head body lengths of 43.34﹣47.12 mm and forearm lengths of 34.37﹣37.87 mm (Table 2). Their tragus length is less than half of the length of the ear (Fig. 1a). Fur is soft and thick, with dark brown to black hair on the back and gray to dark gray hair on the belly (Fig. 1b, c). Hind-foot lengths of 8.22﹣9.49 mm, more than half of the tibia lengths of 16.08﹣17.23 mm, respectively (Table 2). Interfemoral and wing membranes are brown, while the wing membrane is attached to the metatarsal bone below the ankle joint (Fig. 1d). Skull is delicate, with a greatest length of 14.92﹣15.82 mm and a breadth of braincase 7.46﹣8.04 mm (Table 2). A noticeable inclination was observed at the frontal bone, brain is slightly enlarged and round and higher than the maxilla. Their skulls are flat and the zygomatic arch is thin. The external and craniodental characteristics are consistent with those of Myotis horsfieldii. Phylogenetic analysis using the cytochrome b (Cyt b) gene sequence revealed that the bats collected in this study clustered well with M. horsfieldii previously collected in Laos, Malaysia and Hong Kong, China (Fig. 2). The genetic distance was less than 0.8% compared to the specimen from Hong Kong, China (Table 3). Historically, the presence of M. horsfieldii was recorded in regions such as Jiangxi, Guangdong, Hainan, Hong Kong, and Hunan. Our findings augment the known geographical distribution of this species (Fig. 4), establish its novel presence in the provinces of Zhejiang and Anhui. Presently, the M. horsfieldii specimens are deposited in Key Laboratory of Conservation and Application in Biodiversity of South China, Guangzhou University, and these specimens are numbered GZHU 23118, GZHU 23119, GZHU 23642﹣GZHU 23646, GZHU ah23013, GZHU 23047, and GZHU ah23048.