Trypanosoma cruzi, the hemoflagellate parasite and cause of Chagas disease in Latin America, is carried by Triatomine vectors, principally Triatoma dimidiata and Rhodnius prolixus in Central America. To assist control efforts and to understand the epidemiology of the disease in Guatemala, the population genetics of T. dimidiata was analyzed among three houses within a village and two adjacent villages in Guatemala. Eleven Randomly Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR) primers were screened and three used to amplify bands, 29 of which were scored, from T. dimidiata DNA of ≈50 bugs per house from three houses within a village and from 66 and 33 bugs, respectively, from adjacent villages. Results show very small genetic distances among the three T. dimidiata subpopulations from the houses (D = 0.013–0.022) and the two villages (D = 0.0199). The amount of differentiation among houses (fixation index, F_ST) was also very small, F_ST = 0.025 among the houses and the two villages F_ST = 0.019. These fixation indices give an average number of mating migrants per generation (Nm) of 9.7 (among houses) and 12 (among villages). Average heterozygosity (H) appears to be high, ranging from H = 0.299–0.325 among the houses and H = 0.273 among the villages. The low genetic distance and fixation indices, and high heterozygosity suggest that the subpopulations in the houses and in the adjacent villages are not reproductively isolated but are in fact, one large panmictic population. Therefore the geographic coverage necessary for effective control must include, at least, the area encompassing adjacent villages.