Leishmania chagasi, the agent of visceral leishmaniasis in dogs in the Americas has a tropism to the male genital system, particularly the epididymis, prepuce, and glans penis, resulting in shedding of Leishmania in the semen. The goal of this study was to verify the possibility of venereal transmission of L. chagasi. Twelve Leishmania-free bitches, housed in the absence of the insect vector, copulated with multiple naturally infected dogs that were shedding Leishmania in the semen. PCR analysis of serially collected ejaculates indicated that shedding of Leishmania in the semen is intermittent. Three bitches seroconverted, and six were PCR positive by the end of the experimental period (165 days after the last copulation). These data support the notion that L. chagasi may be sexually transmitted from naturally infected dogs to susceptible bitches in the absence of the biological insect vector.