PCR flowcharts and satellite-DNA sequencing were used to genotype Trypanosoma cruzi. Parasitic load of chronic patients was limiting for flowcharts but not for satellite-DNA sequencing. Most DTUs identified belonged to samples from neonates and pediatric cases. The most identified DTU was TcV, common in Bolivian domestic cycles. A mixed infection TcV–TcII was detected for the first time simultaneously in Bolivian migrants.