Leptospirosis has been identified as a zoonotic disease caused by pathogenic spirochetes of the bacterial genusLeptospira. Rodents are considered the primary hosts of these bacteria, whereas many recent studies suggest that bats may serve as potential natural reservoirs. However, studies on pathogenic spirochetes hosted by bat populations still need to be completed in China. In this study, a total of 276 bats belonging to five genera collected in Yunnan Province (Southwest China) from 2017 to 2021 were included in the screening. Pathogenic spirochetes were detected by PCR amplification and sequencing targeting four genes (rrs,secY,flaB,andLipL32), resulting in 17 positive samples. Phylogenetic analysis based on multi-loci concatenated sequences, inferred by MLST approach, identified the strains as two novelLeptospiraspecies within the pathogenic group. Of note, onlyRousettus leschenaultiiwas found to harbor these spirochetes, suggesting it may be one of the potential natural reservoirs in circulating leptospires in this region. Nevertheless, the pathogenesis and transmission dynamics still need to be fully understood, requiring in-depth studies on other animals and the surrounding population.