Trypanosoma cruzi presents considerable genetic and protein profile polymorphism among different parasite populations. In a previous work, our group indicated cysteine and metalloprotease activities as good markers for separating T. cruzi I (TCI) from T. cruzi II (TCII) isolates, with higher heterogeneity among TCII isolates. Here, we have investigated the expression level of surface cruzipain in 16 field isolates belonging to the genotype TCI (n = 8) and TCII (n = 8) of T. cruzi. By means of flow cytometry analyses, using an anti-cruzipain polyclonal antibody, we observed a highly heterogeneous pattern of surface cruzipain molecules in these isolates, independently of their genotypes, cell measurements (size and granularity), original hosts, or biomes. However, fluorescence labeling tended to be stronger in TCI than in TCII population. Interestingly, isolates that expressed higher levels of surface cruzipain also yielded elevated levels of metacyclogenesis in vitro.