Multilocus enzyme electrophoresis is the tool most frequently used to classify Leishmania spp., although it is time consuming and, sometimes, a not enough discriminative method. In the present study a kinetoplast DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to characterize 16 zymodeme MON-1 Leishmania infantum strains: 15 were from dogs housed in public kennels of 7 geographical areas in the Emilia-Romagna region, Northern Italy, 1 was the L. infantum reference strain MHOM/TN/1980/IPT1. Six enzymatic patterns were observed. Kinetoplast DNA RFLP-PCR confirmed to have a good discriminatory power within the same zymodeme and proved to be useful for comparing few strains or discriminating between relapse and reinfection in the same host. We therefore recommend it use for discriminating between relapse and reinfection in the same host rather than supporting large-scale epidemiological studies.