The phlebotomine sand fly Lutzomyia verrucarum is the vector of human bartonellosis in Peru. It is found associated with human habitation at elevations between 1,000-3,000 m in 3 major valleys separated by the Andean mountain ranges. Populations within valleys were predicted to be panmictic and demonstrating varying degrees of genetic isolation between valleys. The genetic structure of L. verrucarum was examined by amplification of a fragment of the mitochondrial genome that included the NADH dehydrogenase subunit 4 (ND4), ND4L, ND6 and cytochrome b (cytb). Specific primers pairs were selected to amplify 1,400 bp of the ND4, 700 bp of cytb and a 360 bp fragment of 12S rDNA. The amplicons were characterized by SSCP (single strand conformation polymorphism) and 3 representatives of each unique SSCP profile were sequenced. Sequences of ND4, cytb and 12S rDNA were obtained from 30 specimens collected from villages representing the 3 valleys. Specimens from Lima (coastal Peru) and Huanuco (eastern Peru) provided outgroup comparators. 12S rDNA sequences appeared to be less informative, with less than 0.3% (~1 bp)difference between sequences from the most distantly separated specimens. Cytb and ND4 sequences demonstrated a higher level of differentiation. When analyzed phylogenetically, cytb sequences from samples collected in central Peru clustered in a lineage distinct from that of Lima and Huanuco. The data indicate that genetic structuring has occurred within the 1,200 x 300 km geographic range of L. verrucarum.