Molecular analysis of Shiga toxin-producing Escherichia coli (STEC) from different sources is considered as a major approach to assess their risk potential. However, only limited data are available about the correlation of evolutionary relationship, the presence of major virulence factor genes and the putative risk of an STEC strain for human infection. In this study, we analysed the evolutionary relationship of 136 pathogenic E. coli strains from human, animal and food sources by multi-locus sequence typing (MLST) and molecular subtyping of their Shiga toxin (stx) and intimin (eae) genes. Moreover, the distribution of three type III effector genes, encoded within the locus of enterocyte effacement (LEE), and 16 effector genes, which are encoded outside the LEE, was analysed. One hundred and five strains from different sources harboured 5–15 of the analysed non-LEE-encoded effector genes. In 101 of these strains, the LEE genes eae, map, espF and espG were present simultaneously. Thirty-one isolates deriving mainly from food and patients suffering from haemolytic uraemic syndrome (HUS) were eae-negative and did not carry any of the analysed effector genes. By combination of MLST and virulence gene data, we defined five genetic clusters. Within these clusters a clear-cut affiliation of particular sequence types and the occurrence of certain effector genes was observed. However, in contrast to other studies, a significant correlation between the amount and type of effector genes and the risk to cause HUS could not be demonstrated.