Enterotoxigenic Escherichia coli (ETEC) can be attributed to around 200 million diarrheal episodes and 380,000 deaths in the developing regions. Travelers' diarrhea occurs in 15-40% of travelers to developing regions with ETEC being the most important etiologic agent. This study aims to describe the distribution of enterotoxins and colonization factor (CF) profiles of ETEC isolates from stool samples of adult travelers acquiring diarrhea in Mexico, Guatemala, and India and a group of children with acute diarrhea in Houston, TX, between 2007 and 2012. The heat-labile/heat-stable (LT/ST) enterotoxins and CFs from 252 patients were determined using polymerase chain reaction assay. Among the 252 ETEC isolates, 15% were LT-only, 58% were ST-only, and 28% produced both LT and ST. The distribution of LT-only (12-15%) and ST-only (55-56%) isolates was similar between Latin American and Indian sites. The most prevalent CF was CS21, expressed in 65% of the isolates followed by CS6 (25%) and CS3 (17%). Among the international travelers, 64% of the ETEC isolates expressed CS21. CS21 was expressed in 46% of isolates from Latin America compared with 96% of isolates from India (P < 0.0001). CS21 was expressed in 85% isolates from Houston children. CS21 was increasingly found in ST-only (P = 0.003) and ST/LT (P = 0.026) ETEC compared with LT-only ETEC. High frequency of finding CS21 among recent isolates of ETEC over a wide geographic distribution warrants additional studies on this CF. Highly conserved CS21 is an important target for potential multivalent ETEC vaccines.