ABSTRACT The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtyping of virulence factors and markers [ stx1 , stx 2a / stx 2c , q21 / Q933 , tir ( A255T ), and rhsA ( C3468G )]. LSPA6 lineage II dominated among bovine isolates (63%), followed by lineage I/II (35.6%) and lineage I (1.4%). In contrast, the majority of the human isolates were typed as lineage I/II (77.6%), followed by lineage I (14.1%) and lineage II (8.2%). Multivariate analysis revealed that the tir ( A255T ) SNP and the stx 2a /stx 2c gene variants were the genetic features most differentiating human from bovine isolates. Bovine and food isolates were dominated by stx 2c (86.4% and 65.5%, respectively). Among human isolates, the frequency of stx 2c was 36.5%, while the frequencies of stx 2a and stx 2a plus stx 2c were 41.2% and 22.4%, respectively. Bovine isolates showed equal distribution of tir (255A) (54.8%) and tir (255T) (45.2%), while human isolates were dominated by the tir (255T) genotype (92.9%). LSPA6 lineage I isolates were all genotype stx 2c and tir (255T), while LSPA6 lineage II was dominated by tir (255A) (86.4%) and stx 2c (90.9%). LSPA6 lineage I/II isolates were all genotype tir (255T) but showed more variation in stx 2 types. The results support the hypothesis that in The Netherlands, the genotypes primarily associated with human disease form a minor subpopulation in the bovine reservoir. Comparison with published data revealed that the distribution of LSPA6 lineages among bovine and human clinical isolates differs considerably between The Netherlands and North America.