In this study, we report on the prevalence of 19 virulence genes in enteroaggregative Escherichia coli (EAEC) isolates from northern South Africa. Stool samples obtained prospectively from 97 children from 1 to 12 months of age were analyzed, and EAEC isolates were confirmed based on the presence of aaiC or aatA genes. We investigated 177 enteroaggregative Escherichia coli isolates for the prevalence of virulence genes using multiplex polymerase chain reaction. The chromosomal gene aaiC was detected at higher frequency (48.0%) compared with aatA (26.0%). The gene encoding the open reading frame Orf61 was the most prevalent putative virulence trait detected among the isolates (150/177; 84.7%). None of the genes was statistically associated with diarrhea (P > 0.05). Detection rates were higher during 7-12 month of life with an association observed for the pic gene and the age group 7-12 months (P = 0.04). Winter was the season with the highest detection rates. Our data reveal a high prevalence of Orf61, Orf3, and astA in South African EAEC isolates. Specific genes may provide additional markers for the study of disease associations with age and season of sample collection.