Trypanosoma cruzi is genetically classified into at least two major lineages named T. cruzi I (also named Tc I) and T. cruzi II (also named Tc IIb). T. cruzi II is associated with Chagas' disease in the southern cone of South America, while T. cruzi I is the only one so far identified in chagasic patients of Central America and in the northern part of South America. Herein we identified T. cruzi IIb directly in 9.9% of blood of chronic chagasic patients of Colombia. This finding establishes that in this region, the two T. cruzi lineages are associated with the pathology of Chagas' disease and have implications in the morbidity and epidemiology of the disease.