Randomly amplified polymorphic DNA (RAPD) technique was used to study the genetic structure of sylvatic, peridomestic and domestic populations of Triatoma dimidiata. The genetic flow among them was calculated to establish the epidemiological risk of non-domiciliated populations in the transmission of Chagas disease in an endemic area of Boyaca, Colombia. A total of 83 adult specimens were studied: 26 sylvatic, 27 peridomestic and 30 domestic insects. Wright's Fst was 0.071 and the effective migration rate (Nm) 3.3, suggestive of low genetic differentiation and a movement of at least three insects per generation. The calculated percentage of polymorphic loci was 99%, confirming a large average heterozygosity due to a permanent contact between insects of the three populations. These results imply that non-domiciliated populations of T. dimidiata represent an epidemiological risk in the transmission of Chagas disease owing to the fact that they can colonize human dwellings. Close surveillance of non-strictly domiciliated species of triatomines such as T. dimidiata should entail not only the domicile but also the peridomicile and should include control programs of animal reservoirs. Houses enhancement, educational programs, surveillance of reinfestation and of individuals at risk of infection should be priorities in the control policies in endemic regions such as Boavita, Boyaca.