Sierra Nevada of Santa Marta is one of the most endemic regions of Chagas disease in Colombia. In this study, we compared the biological behavior and genetic features of Trypanosoma cruzi stocks that were isolated from domestic and sylvatic insects in this area. Rhodnius prolixus (from domestic environments) and Triatoma dimidiata (from sylvatic, peridomestic and domestic environments) are the most important vectors in this region. Genetic characterization showed that all stocks corresponded to T. cruzi I, but LSSP-PCR analyses indicated that some genotypes were present in both environments. Biological characterization in vitro showed a low growth rate in sylvatic T. cruzi stocks and in some domestic T. cruzi stocks, possibly indicating the presence of stocks with similar behavior in both transmission cycles. In parallel, in vivo behavioral analysis also indicated that T. cruzi stocks are variable and this species did not show a correlation between the environments where they were isolated. In addition, all stocks demonstrated a low mortality rate and histopathological lesions in heart, skeletal muscle and colon tissue. Moreover, our data indicated that experimentally infected chagasic mice displayed a relation between their myocardial inflammation intensity, parasitism tissue and parasite load using the qPCR. In conclusion, our results indicate that the T. cruzi stocks present in SNSM have similar biological behavior and do not show a correlation with the different transmission cycles. This could be explained by the complex transmission dynamics of T. cruzi in Sierra Nevada of Santa Marta, where hosts, vectors (e.g., T. dimidiata) and reservoirs circulate in both environments due to the close contact between the two transmission cycles, favoring environment overlapping. This knowledge is an important key to understanding the epidemiology and pathology of Chagas disease in this Colombian region. Furthermore, our findings could be of significant use in the design of control strategies restricted to a specified endemic region.