The population genetics of Triatoma dimidiata (Latreille, 1811) from five different provinces in Guatemala, including three sylvan and three domestic populations, was investigated by random amplification of polymorphic DNA-polymerase chain reaction. There is a high degree of genetic variation in all of the T. dimidiata populations as evidenced by high levels of average expected heterozygosity and polymorphism. Domestic populations are more closely related to each other (D = 0.05–0.085, Nei’s genetic distance) than are the sylvan (D = 0.121–0.189). Within the limited sample size of three populations, there was a correlation with geographic and genetic distance for the domestic populations, but not for the sylvan. Surprisingly, one of the sylvan populations was genetically very similar to the domestic populations. The F_ST demonstrated a high degree of differentiation at the country-wide level (F_ST = 0.175) and a moderate degree of differentiation within the sylvan (F_ST = 0.135) or domestic (F_ST = 0.097) populations. Although these results demonstrated that gene flow is limited between different provinces in Guatemala, hierarchical analysis showed that barriers between the Atlantic and Pacific drainage slopes were not biologically significant limiters of gene flow.