SUMMARY Using the polymerase chain reaction (PCR) test for diagnosis of canine leishmaniasis has greater sensitivity and specificity than culture and visualization of the parasite. This study compares PCR for the diagnosis of the genus and species of Leishmania with serological techniques used for the control of canine visceral leishmaniasis (CVL) in Brazil, considering two regions. We analysed peripheral blood samples collected from 195 dogs in the Campinas (SP) and Teresina (PI) regions. ELISA was performed as a serological method and PCR was performed using specific primers for the genus Leishmania spp. and the species Leishmania chagasi. In Campinas, a greater sensitivity of PCR (88.24%) (P = 0.0455) compared to Teresina (14.71%) (P < 0.0001) was observed, and an agreement was observed for Cohen's kappa index (0.9096). Both PCR and ELISA showed discordance for sensitivity (Campinas 100%, Teresina 21.74%), specificity (Campinas 30.77%, Teresina 100%), positive predictive value (Campinas 68.97%, Teresina 100%), negative predictive value (Campinas 100%, Teresina 37.94%) and Cohen's kappa index (0.1238). This study confirms the importance of PCR in analysis of the canine reservoir, and as an effective method for the detection of active and recent infection.