Enteroaggregative Escherichia coli (EAEC) strains have been implicated as emerging aetiological agents of diarrhoea worldwide. In the present study, 43 EAEC strains were serotyped and characterized according to random amplification of polymorphic DNA profiles, PFGE, multilocus enzyme electrophoresis (MLEE) and the presence of putative virulence genes (hly, aero, kps, fim, aggA, aafA, aggR, astA, she, aap, shf and pet). The EAEC strains consisted of a diversity of serotypes including eight O-non-typable and 35 O-typable strains arranged into 21 O:H combinations. Amplification of specific genes revealed that all strains carried at least two of the virulence sequences investigated. fim, aggR and aap were the most frequent genes in both groups studied. hly, aero and aggA sequences were more prevalent in the diarrhoeal group. kps occurred exclusively in strains isolated from symptomatic children and showed strong association with diarrhoeal disease. The molecular approaches used to investigate the relatedness among EAEC strains revealed a high degree of polymorphism, suggesting that these micro-organisms have a non-clonal origin. A closer relationship was observed among EAEC strains sharing O:H types. No significant clustering could be identified related to the virulence traits investigated; however, the she locus showed clonal distribution by MLEE typing. These results are in accordance with previous findings in revealing the conservation of particular EAEC factors, despite the high degree of diversity related to both genotypic and phenotypic markers.