Human and canine visceral leishmaniasis caused by Leishmania infantum emerged in central Israel after an absence of over 30 years. The origin of this outbreak was investigated by examining genetic polymorphisms in 37 strains isolated from dogs and patients with visceral leishmaniasis in the continuously active northern Israeli and West Bank foci and in a new Israeli focus using DNA fingerprinting with the human multilocus minisatellite probe 33.15. Analysis of the patterns obtained by DNA fingerprinting separated the strains geographically into northern (clade B) and central (clades A and C) genotypic groups. These results suggest that the emergence of visceral leishmaniasis in central Israel is due not to parasite spread from northern Israel to the new focus but rather to increased parasite transmission in central Israel and the West Bank coupled with changes in the ecoepidemiology of this region.