Enteroaggregative E. coli strains are important causes of diarrhea worldwide and are the second most important bacterial cause of travelers' diarrhea (TD). Pathogenicity of EAEC is not completely understood. We investigated the occurrence of putative virulence related genes (VRG), aatA, aggR and aaiC, in a nested case-control study of a cohort of US travelers >18 years of age, visited either Guatemala or Mexico. Fecal samples were collected between 2008 and 2012 from patients with TD from whom a HEp-2 cell adherent EAEC strain was identified (Cases) and from healthy subjects in the same locale without diarrhea from whom enteric pathogens were not isolated (Controls). Thirty-one subjects with acquired TD at destination was compared with 32 healthy controls. aaiC was the most expressed virulence related gene in 21 (67.7%) cases vs. 2 (6.3%) controls, (P < 0.000). aggR was found in 18 (58.1%) cases vs. 1 (3.1%) control, (P < 0.000). aatA in 9 (29.0%) cases vs. 1 (3.1%) control (P < 0.006). With genes combined, aaiC+aggR were seen together in 18 (58.1%) cases vs. 1 (3.1%) control (P < 0.000); aaiC+aatA were identified in 9 (29.0%) cases vs. 1 (3.1%) control (P < 0.006); aggR+aatA were present in 9 (29.0%) cases vs. 1 (3.1%) control, (P < 0.006). All three putative genes, aaiC+aggR+aatA were found in 9 (29.0%) cases vs. 1 (3.1%) control, (P < 0.006). The PCR products showed that aaiC, aggR, and aatA occurred in higher frequency and were more commonly associated with EAEC in cases of TD acquired in the two countries of study, as compared to controls. aaiC was found in all cases from Guatemala. Further research is needed to study geographic and host factors in EAEC-causing travelers' diarrhea.