Trypanosoma cruzi possesses hundreds of genes associated with pathogenesis. The extent and organization of this diverse gene repertoire, expression, and role in infection remain unclear. Using accurate long-read sequencing and chromatin conformation capture, we assembled T. cruzi Sylvio X10 strain chromosomes from telomere-to-telomere. The genome provides accurate organization of multigene family genes, confirming their distribution in expanded clusters or scattered throughout the chromosomes. Quantitative proteomics shows stage-specific proteins and numerous trans-sialidases upregulated in trypomastigotes. The expression of virulence gene families varied in trypomastigotes after each round of cell infection, resulting in heterogeneous parasite populations with variable cell invasion capacity. A T. cruzi genome-wide yeast surface display screen against Chagas disease patients’ antibodies reveals genes expressed during human infections. However, limited conservation in their antibody-binding sites suggests their sequence diversity and variation might help parasites avert antibody recognition. The data point to a role for some multigene families in infection persistence.