Serology, bone marrow (BM)-, lymph node (LN)- and whole blood-polymerase chain reaction (PCR) were evaluated as potential reference tests for the diagnosis of canine leishmaniasis. A high degree of agreement (91.0%) was observed between Leishmania cultures and serology or BM/LN-PCR. In the light of these results as well as the access to biological test material and the cost of each method, LN-PCR is recommended for the diagnosis or therapeutic control of canine leishmaniasis, but BM-PCR is a suitable alternative in dogs without detectable adenomegaly. For large-scale epidemiological field studies, antibody detection is appropriate and whole blood-PCR can be used to complement the serological results.