The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.