To further study genetic heterogeneity of Mexican stocks of Trypanosoma cruzi, genomic Southern analyses from 54 Mexican isolates and 5 South American reference stocks were carried out. The membranes were hybridized with a homologous cDNA clone from the ribosomal protein S4 that identifies allelic bands from a single gene type locus. These allelic bands were sequentially numbered depending on their relative size. Mexican T. cruzi stocks were quite homogeneous: 31 cases (57%) showed a homozygous genotype 3/3, and 21 isolates (38%) exhibited the heterozygote genotype 2/3. Just 2 Mexican stocks (3%) showed a different genotype 2/5, but the potential parental homozygous 2/2 was never observed. Being that T. cruzi is a diploid organism, the apparent absence of the presumptive parental homozygous genotype 2/2 argues against sexual reproduction within the population, at least as a common event. Therefore, these data support a clonal population structure of T. cruzi in Mexico.