Electrophoretic studies of Guatemalan blackflies, Simulium ochraceum, the main vector of Central American onchocerciasis, were performed using isozyme patterns. A total of six larval and adult populations originating from four localities (a non-endemic area, Finca La Ruda, a low-endemic area, Finca Rincon, and two high-endemic areas, Finca Brote and Finca Recreo) were analyzed. The enzymes examined were adenylate kinase (AK), alkaline phosphatase (ALP), glucose phosphate isomerase (GPI), hexokinase (HK), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malic enzyme (ME), and phosphoglucomutase (PGM). Of these, six enzymes, AK, ALP, HK, IDH, LAP, and ME showed no variation either within or among populations. GPI and PGM were found to be highly polymorphic in all of the six populations. χ2-test for fit to Hardy-Weinberg equilibrium (HWE) revealed that the distribution of the phenotypes of the two polymorphic enzymes was not significantly out of the HWE. A verages of values of proportion of polymorphic loci (P) and heterozygosity per individual (H) of the six populations were 0.234 and 0.084, respectively, each of which was similar to the values reported for different groups of organisms (Nei, 1975). Genetic diversity among populations was measured by FST and GST and both parameters showed that the non-endemic population from the Finca La Ruda (Dl) was greatly differentiated genetically from the other five populations. Estimates of genetic distance (Nei, 1975) showed that the Dl population was furthest from the other populations.