Bovine leptospirosis is one of the most important reproductive diseases that compromise the productivity of cattle farming. However, the presence of the agent on vaginal environment is still poorly understood in cattle. Considering this context, the present study aimed to detect the presence of pathogenic Leptospira sp. in vaginal fluid (VF) of cows. VF and urine were collected from 254 cows from a slaughterhouse for bacteriological culture and PCR (lipL32 gene). Overall, eleven pure culture (4.3%) of leptospiral isolates were obtained. Leptospiral DNA was detected in 128 (50.4%) of VF samples and 81 (31.0%) of urine samples, while on 75 (29.5%) it was exclusively in VF and 28 (11.3%) only in the urine. Detection of leptospiral DNA and the recovery of viable leptospires from VF of a high number of cows without apparent symptoms highlight the role of vaginal carriers and indicate that venereal transmission (female-to-male) could occur in that species. Moreover, VF should be encouraged as a valuable sample for diagnosis of bovine genital leptospirosis.