Traveller's diarrhoea (TD) is the most common illness reported in international travellers. TD is caused by a wide range of pathogens, including bacteria, viruses and parasites. Multiplex PCR assays can be especially useful for studying the aetiology of TD. The first objective of this study was to evaluate the utility of the commercially available multiplex PCR (xTAG® Gastrointestinal Pathogen Panel (GPP)) for the diagnosis of TD. A total of 185 stool specimens obtained from 174 patients were processed using the GPP assay. This test detected 86 pathogens in 67 stool samples (67/185, 36.2%). Sixteen pathogens out of 86 were also detected by routine testing. The remaining pathogens (n = 70) required further confirmation by alternative techniques. Finally, 60 out of 70 pathogens were confirmed. The second objective of this study was to analyse the aetiology of TD based on the results obtained by the GPP test and routine methods. The primary pathogens causing TD were Shigella (24.2%) followed by enterotoxigenic Escherichia coli (ETEC) (23.2%), enteroaggregative E. coli (14.7%) and Giardia (13.7%). Significant regional differences were observed for ETEC with 19.4% of TD cases acquired in Africa, 11.3% in Asia and none in South Central (SC) America (p 0.01), Giardia was found in 1.5% of cases among those who had travelled to Africa, 14.1% of those who had travelled to Asia and 3% of those who had travelled to SC America (p 0.01). In conclusion, the GPP test improved the detection of enteropathogens and allowed better assessment of the aetiology of TD.