Bats from three provinces in Vietnam (Lai Chau, Son La, and Dong Thap) were examined for the presence of pathogenic Leptospira or specific antibodies using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and microscopic agglutination test (MAT). Tissue specimens from 298 bats belonging to 11 species were analyzed using a real-time PCR assay specific for leptospires of pathogenic species. Leptospiral DNA was identified in 40 bats from following species: Rousettus amplexicaudatus (5/9; 55.5%), Rousettus leschenaultii (17/42; 40.4%), Myotis hasseltii (8/25; 32%), Taphozous longimanus (3/12; 25%), and Eonycteris spelaea (7/32; 21.9%). Based on secY phylogeny, sequences from M. hasseltii bore a strong resemblance to L. borgpetersenii. Sequences from other species revealed unique lineages: one of them resembled Leptospira sp., previously identified in Rousettus madagascariensis (Madagascar) and Rousettus aegyptiacus (South Africa); the second lineage showed close relation to L. kirshneri; and the third held an intermediary position between L. noguchii and L. interrogans. Through ELISA, anti-Leptospira antibodies were found in 83 of 306 bats, with the highest seroprevalence observed in R. leschenaultii (44/48; 91.6%), R. amplexicaudatus (6/8; 75%), and E. spelaea (19/25; 76%). 66 of these ELISA-positive samples were tested using MAT; 41 of them were confirmed in MAT as positive. The predominant serogroups in our study were Tarassovi and Mini.