鼠耳蝠属（Myotis）是陆生脊椎动物中分布最广泛的类群之一，全世界共140种，中国29种，而且随着分类学研究的深入，该属内新的种类还在不断被发现。本研究基于经典分类学（包括外部形态特征、毛色及头骨等特征比较）、辅以分子生物学研究手段分别分析了采集于云南双柏（9号标本）和永德（2号标本）的标本。结果显示：上述标本在分类上均属于鼠耳蝠属，但不属于现有已描记的任何鼠耳蝠物种，而且遗传距离上已达到种级分化水平。据此，本研究将这两个类群分别描述为新种：双柏鼠耳蝠 Myotis shuangbaiensis Li & Qian，sp. nov.及永德鼠耳蝠 Myotis yongdensis Li & Qian，sp. nov.。双柏鼠耳蝠的鉴别特征为背毛基部黑色、毛尖浅棕，腹毛毛基黑色、毛尖灰白，翼膜附着于后足第一趾基部，上内门齿（I²）与上外门齿（I³）大小相近，第二下前臼齿（P₂）齿高小于下犬齿之半，第三下前臼齿（P₃）的齿基面积和齿高均分别约为第二下前臼齿（P₂）的三分之二。永德鼠耳蝠的鉴别特征为背毛深棕红色，腹毛基部棕黑色、毛尖灰白色，股间膜腹面具密度适中的短毛，翼膜附着于后足跖骨基部，耳屏顶端圆弧形，第三上前臼齿（P³）的齿高约为第二上前臼齿（P²）之半。本研究结果不仅反映了鼠耳蝠属内的分类学研究仍存在诸多的未知领域，而且还进一步增添了中国西南地区生物多样性的丰富度。

The genus Myotis is one of the most widely distributed genera of terrestrial vertebrates. It includes 140 recognized species worldwide, 29 of which are recorded in China. Ongoing taxonomic research continues to uncover new species within this group. In this study, we employed classical taxonomic approaches, including comparisons of external morphology and cranial characteristics, complemented by molecular biological methods to analyze bats collected from Shuangbai (n = 9) and Yongde (n = 2) in Yunnan Province. The results show that these specimens belong to Myotis, but do not belong to any of the existing Myotis species that have been described, and have reached the genetic distance of species-level differentiation. Therefore, we describe them as two new species: Myotis shuangbaiensis Li & Qian, sp. nov. and Myotis yongdensis Li & Qian, sp. nov. The diagnostic features of Myotis shuangbaiensis include dorsal fur with black bases and light brown tips, and ventral fur with black bases and grayish-white tips. The plagiopatagium attaches to the base of the first toe on the hindfoot. The upper inner incisor (I²) and upper outer incisor (I³) are similar in size. The crown height of the second lower premolar (P₂) is less than half that of the lower canine. Both the basal area and crown height of the third lower premolar (P₃) are approximately two‑thirds those of the second lower premolar (P₂). The diagnostic features of Myotis yongdensis include dark reddish‑brown dorsal fur, and ventral fur with brownish‑black bases and grayish‑white tips. The ventral side of the interfemoral membrane is covered with moderately dense short fur. The plagiopatagium attaches to the base of the metatarsus of the hindfoot. The tragus has a rounded tip. The crown height of the third upper premolar (P³) is approximately half that of the second upper premolar (P²). These findings reveal significant persistent knowledge gaps in Myotis taxonomy and enhance the understanding of biodiversity richness in Southwestern China.