The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil once the dog is the main reservoir host of the disease. The aim of this study was to evaluate the conjunctival swab (CS) as a mass-screening tool for CVL molecular diagnosis in an endemic area classified as priority for the Brazilian Ministry of Healthy for surveillance action. A total of 1350 domiciled dogs were screened. The animals were evaluated by serological tests (enzyme-linked immunosorbent assay (ELISA) as screening and immunofluorescence antibody test (IFAT) for confirmation) and by CS associated to real-time PCR, using primers addressed to kinetoplast DNA (kDNA) minicircles and SYBR Green. Canine β-globin gene amplification was used to evaluate the sample DNA integrity. A subgroup of 484 animals was also submitted to clinical evaluation. Among the 1350 dogs screened, 369 (27.3%) were positive by CS real-time PCR and 126 (9.3%) tested positive by ELISA. Thirty-one percent (39/126) of the ELISA-positive dogs were confirmed by IFAT. CS real-time PCR was able to detect infection in dogs independently of the symptomatology degree (p > 0.05), while ELISA was more sensitive in the group of dogs that present three or more clinical signs related to CVL. The results demonstrated that CS real-time PCR was able to detect a higher number of infected dogs than ELISA and that the prevalence of canine infections has been underestimated by the serological assays. The use of sensitive molecular diagnostic methods like CS real-time PCR, mainly in endemic areas, could greatly contribute to disease control.