In this article, we describe the findings obtained using immunoblot analysis in the diagnosis of canine visceral leishmaniasis (VL) and its correlation with serological titer and clinical status. We found that all animals bearing amastigote forms recognized antigens with 29 and 32 kDa and that this pattern can be exploited for diagnostic and epidemiological purposes. The recognition of the 29- and 32-kDa antigens was verified even in seronegative dogs and preceded seroconversion in periods ranging from several months to 2 yr. We found a correlation between serological titer and parasite burden. Although no correlation between antigenic recognition pattern and clinical status was observed, immunoblot analysis proved to be a reliable test to detect antibodies against Leishmania sp. antigens in dogs from areas with endemic VL.