Naturally Leishmania infantum infected bitches were divided into oestrogenized (n = 11) and non-oestrogenized (n = 6) groups. Vaginal secretions were collected for polymerase chain reaction (PCR), and vulval, vaginal and uterine tissues were collected for the immunohistochemical (IHQ) identification of L. infantum. Parasite DNA was identified in vaginal secretions of non-oestrogenized (41.8%) and oestrogenized (18.2%) bitches (P<0.05; Fisher's Exact test). IHQ was positive in vulvar dermis (23.5%) and vaginal mucosa (17.7%) but negative in endometrium of all bitches. Poor association between positive vaginal secretion PCR and tissue IHQ (Kappa index) were observed. The results showed that genital secretions are a potential source for dog contamination.