Background: We studied geographic distribution of diarrhoeagenic Escherichia coli virulence genes (DEC VGs) acquisition in travellers and investigated if they acquired highly virulent EAEC/STEC hybrid strains. Methods: From the prospective, multicentre COMBAT study among 2001 Dutch travellers, 491 travellers were selected based on travel destination to 7 subregions. Faecal samples taken directly before and after travel were screened for nine DEC VGs with real-time PCR. Incidence proportions and rates were calculated for each gene and subregion. Results: 479 travellers were analysed. 21.8% acquired aggR (EAEC), with highest acquisition rates in Northern and Western Africa and 15.3% acquired eae (STEC/EPEC) with highest rates in travellers to Western and Eastern Africa. ETEC (elt or est gene) was acquired by 4.2% of travellers and acquisition of est was associated with traveller's diarrhoea. Overall, the risk of acquiring DEC VGs was low in Southern Africa and South America. Although the combination of aggR (EAEC) and stx1/2 (STEC) was acquired by 3 travellers, these genes could not be detected together in a single E. coli strain. Conclusions: The risk of acquisition of DEC VGs strongly depends on the travel destination, with those travelling to Africa - except Southern Africa - having a higher risk.