Chagas disease (CD) caused by Trypanosoma cruzi, is a major health problem in Bolivia. In this study, we have assessed the prevalence and the risk of infection by T. cruzi in nine Bolivian departments. The overall average seroprevalence was 19.5% (2-32%) being higher in women between 11-20 years old. The T.cruzi genetic lineages infecting humans was 43.4% TcI; 45.4% TcV, 8.5% TcII and 2.7% for TcV and TcII. T. cruzi infection was detected in 49.1% of the 1,671 Triatoma infestans captured within houses, the majority (65%) made of bricks. Among these, 39.5% were TcI, 52.1% TcV, and 8.4% TcII. The assessment on the knowledge about the disease and the etiological agent revealed that 57% of teachers and health workers are aware of the parasite and 64% of the students receive information about CD, among which, 82% were able to recognize triatomines.