Leishmania infantum polymorphism was studied by DNA microsatellite analysis of 110 L. infantum stocks (94 from dogs, 15 from sand flies, and 1 from a human visceral case) from a rural leishmaniasis-endemic area (Priorat) in northeastern Spain. Three microsatellites of the eight present in three fragments (internal transcribed spacer, Lm4, and Lm2) of L. infantum nuclear DNA are polymorphic inside the focus, resulting in 17 genotypes. Isolates from dogs and sand flies had different allelic compositions and shared only four genotypes. Microsatellite analysis is useful for L. infantum genotyping and epidemiologic tracking. Its application with strains from dogs and vectors in an area endemic for leishmaniasis shows the heterogeneous distribution of L. infantum in hosts living in sympatric conditions.