Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of American visceral leishmaniasis in the New World tropics, has a broad but discontinuous geographical distribution from southern Mexico to Argentina. A baseline for population genetic structure and genetic variability for this species was obtained by analyzing 5 local, peridomestic populations at the approximate center of its distribution, the Magdalena River Valley of central Colombia. Three populations of L. longipalpis from El Callejón, a small rural community, were compared with 2 populations from neighboring areas 12 and 25 km distant for genetic variation at 15 isoenzyme loci. The mean heterozygosity ranged from 11 to 16%, with 1.2 to 2.3 alleles detected per locus. Nei's genetic distances among the populations were very low, ranging from 0.001 to 0.007. Gene flow estimates based on FST indicated high levels of gene flow among local L. longipalpis populations, with minimal population substructuring.