Genetic variability of eight Colombian field populations and two laboratory colonies of a tropical forest sand fly, Lutzomyia shannoni Dyar, was assessed by comparing allozyme frequencies at 20 enzyme loci. Substantial genetic variability was noted in all strains, with mean heterozygosities of 13-21% and alleles per locus of 2.0-2.8. Four loci were monomorphic. Six populations in north and central Colombia showed close genetic similarity (Nei's distances, 0.01-0.09), despite mountainous environment, discontinuous forest habitat, and elevation differences from 125 to 1,220 m. Two samples representing the Orinoco (near Villavicencio) and Amazon (near Leticia) river basins were similar (Nei's distance, 0.08) but diverged substantially from the central six samples (Nei's distances, 0.26-0.40). Although the range of L. shannoni extends from the southeastern United States to northern Argentina, three genetically distinct, geographically discrete, groups were discerned by the current analysis: Orinoco-Amazon river basins, north-central Colombia, and eastern United States.