Brucellosis is a chronic contagious infectious zoonosis that affects the reproductive system of animals, causing economic and health losses. This study diagnosed Brucella spp. in commercial kennels, comparing PCR positivity in different biological samples (blood, semen, and vaginal secretion), as well as correlating these findings with reproductive indices. Hence, we analyzed dogs from kennels in the neighboring cities of Cuiabá and Várzea Grande/MT, Brazil. The reproductive histories of the animals were obtained and blood samples were collected from all animals (n=35); in addition, semen samples were collected from males (n=9) and vaginal swabs were collected from females (n=24) to perform polymerase chain reactions (PCR) for brucellosis. The findings indicated that vaginal swab PCR is an effective test to identify Brucella spp. For males, there were more positive results when testing blood samples, possibly because the male animals were at the beginning stage of infection.