BACKGROUND Chagas disease is a neglected illness caused by the Trypanosoma cruzi parasite, which widely affects American communities. This study attempted to identify T. cruzi genotypes circulating in four indigenous communities of the Sierra Nevada of Santa Marta, Colombia, to investigate parasite transmission dynamics in these communities. In addition, some epidemiological variables to determine the risk factors for infection with this parasite, such as the prevalence of T. cruzi infection, the triatomine species, and the domestic and sylvatic mammals that act as vectors and reservoirs of the parasite in the domestic, peridomestic and sylvatic cycles, were examined. METHODS We developed a prospective study to identify the main risk factors associated with T. cruzi infection in the region. The T. cruzi prevalence was determined by ELISA, IFA and PCR. Triatomines species and both domestic and sylvatic mammals from all communities were captured and sampled. To analyze parasite transmission dynamics in these four communities, eight DNA parasite probes were generated from insect and reservoir samples, and a DNA blot analysis were carried out. RESULTS Serological studies revealed 37% prevalence in the four communities, and Kasakumake was the most endemic region, containing approximately 70% seropositives. Moreover, the molecular diagnosis showed a high correlation between the serological data and the T. cruzi circulating in the patients' blood. A total of 464 triatomine insects were collected in domestic, peridomestic and sylvatic environments, and these insects belonged to five different species; Rhodnius prolixus and Triatoma dimidiata were the two more important species transmitting the parasite. After studying the eco-epidemiological factors in these four communities, the most important risk factors for infection with the parasite were determined. These risk factors are a high infection rate of people and domestic animals, the construction materials of the houses, the presence of infected triatomines inside the human dwellings, the proximity between houses and a sylvatic environment with several triatomine species and wild animals. Finally, the molecular characterization of T. cruzi showed the presence of three haplotypes and complex T. cruzi mixed infections in all reservoirs. CONCLUSIONS Active transmission of T. cruzi is present in four indigenous communities of the Sierra Nevada de Santa Marta with overlap between the domestic and the sylvatic transmission cycles of Chagas disease.