Introduction. Chagas disease caused by Trypanosoma cruzi is one of the most serious public health problems in the Americas. Benznidazole is one of two drugs used to treat Chagas' disease. However, the variation in susceptibility of the parasite to this drug is one of the main causes of treatment failure. Objective. The in vitro susceptibility to benznidazole was assessed in Colombian strains of T. cruzi from several sources and geographical regions. Materials and methods. Thirty-three Colombian T. cruzi strains were isolated from humans, vectors and mammals. These were analyzed in vitro by the MTT enzymatic micromethod to determine the IC50 to benznidazole. Additionally, the in vitro susceptibility was correlated with several biological and eco-epidemiological parameters. Results. Thirty-six percent of the strains were considered to be sensitive, 48% partially resistant, and 16% were resistant. Correlations between the IC50 and several biological and eco-epidemiological parameters indicated that differences in susceptibility depended on the biological source and geographical origin of the strain. Conclusions. A high degree of variability exists in the susceptibility to benznidazole of T. cruzi strains in Colombia. The distribution data indicate the presence and circulation of naturally resistant strains.