In this study, we characterized 272 Shiga toxin-producing Escherichia coli (STEC) isolates from humans, food, and cattle in Belgium [O157 (n = 205), O26 (n = 31), O103 (n = 15), O111 (n = 10), O145 (n = 11)] for their virulence profile, whole genome variations and relationships on different genetic levels. Isolates of O157 displayed a wide variation of stx genotypes, heterogeneously distributed among pulsogroups (80% similarity), but with a concordance at the pulsosubgroup level (90% similarity). Of all serogroups evaluated, the presence of eae was conserved, whereas genes encoded on the large plasmid (ehx, espP, katP) occurred in variable combinations in O26, O103, and O145. The odds of having haemolytic uraemic syndrome was less for all genotypes stx2a, stx2c, stx1/stx2c, and stx1 compared to genotype stx2a/stx2c; and for patients aged >5 years compared to patients aged ⩽5 years. Based on the genetic typing and by using epidemiological data, we could confirm outbreak isolates and suggest epidemiological relationships between some sporadic cases. Undistinguishable pulsotypes or clones with minor genotypic variations were found in humans, food, and cattle in different years, which demonstrated the important role of cattle as a reservoir of STEC O157, and the circulation and persistence of pathogenic clones.