Abstract. The delimitation of cryptic species within the main vector of the American visceral leishmaniasis, Lutzomyia longipalpis, remains a topic of controversy. An analysis of genetic variability based on 8 enzymatic loci revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean heterozygosities that ranged between 6.6% and 6.7%, with 1.6-1.9 alleles detected per locus, while the other had mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3-1.6 alleles per locus. Comparisons of isozyme profiles with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian populations, whereas the other has not been previously reported.Many investigators have suggested that Lutzomyia longipalpis (Lutz and Neiva), the main vector of visceral leishmaniasis in America, is composed of a complex of cryptic species. The evidence published has been nevertheless controversial. Two forms of L. longipalpis were identified from Brazilian colonies that differed in the presence of dorsolateral pale spots in the third and fourth abdominal tergites. 1 Crossing experiments showed a significant reduction in fertility between sympatric and allopatric strains of both forms, but a few fertile hybrids from both sexes were also produced. 1 Because reduced fertility was also recorded between similar individuals from different geographic regions, it was concluded that further field studies should be carried out before these 2 morphologic forms were assigned to specific taxa. Further studies on the distribution of these 2 forms suggested that 1 is found from Mexico to southern Brazil while the other is restricted from northern Brazil to the border with Paraguay. 2 The structure of L. longipalpis as a complex of distinct reproductively isolated populations became more complex as new data on isozymes became available. High genetic divergence, decreased fertility, and the presence of several diagnostic loci between laboratory strains from Costa Rica, Colombia, and Brazil suggested the presence of at least 3 sibling species. 3 However, the adequacy of laboratory colonies to study genetic divergence across geographic areas remains debatable since colonies can be subjected to a significant reduction of genetic variability due to the loss of infrequent alleles and decreased heterozygosity. 4,5 In fact, the detection from field samples of several loci intermediate between diagnostic loci from Colombian and Brazilian colonies suggested that a high polymorphism could also explain the high g...