Cattle comprise a main reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC). The significant differences in host prevalence, transmissibility, and virulence phenotypes among strains from bovine and human sources are of major interest to the public health community and livestock industry. Genomic analysis revealed divergence into three lineages: lineage I and lineage I/II strains are commonly associated with human disease, while lineage II strains are overrepresented in the asymptomatic bovine host reservoir. Growing evidence suggests that genotypic differences between these lineages, such as polymorphisms in Shiga toxin subtypes and synergistically acting virulence factors, are correlated with phenotypic differences in virulence, host ecology, and epidemiology. To assess the genomic plasticity on a genome-wide scale, we have sequenced the whole genome of strain EC869, a bovine-associated E. coli O157:H7 isolate. Comparative phylogenomic analysis of this key isolate enabled us to place accurately bovine lineage II strains within the genetically homogenous E. coli O157:H7 clade. Identification of polymorphic loci that are anchored both in the chromosomal backbone and horizontally acquired regions allowed us to associate bovine genotypes with altered virulence phenotypes and host prevalence. This study catalogued numerous novel lineage II-specific genome signatures, some of which appear to be associated intimately with the altered pathogenic potential and niche adaptation within the bovine rumen. The presented extended list of polymorphic markers is valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies of this emerging human pathogen.Shiga toxin-producing, non-sorbitol-fermenting, and -glucuronidase-negative Escherichia coli (STEC) O157:H7 has evolved from an O55:H7-like progenitor (24, 50, 65) into an emerging human pathogen, with cattle as the main asymptomatic reservoir (12, 54). E. coli O157:H7 is transmitted from cattle to human by means of contaminated food products, such as undercooked meat, unpasteurized milk, fruit and vegetables, or tainted water. As seasonal changes can influence the prevalence and load of E. coli O157:H7 in cattle and super shedders exist within the bovine population (30,33,39,49,67), physical contact of humans with cattle and their environment introduces added risk of E. coli infection (29). Human infection manifests in various ways, ranging from mild to more severe bloody diarrhea. In some cases, infection can lead to renal dysfunction, i.e., hemolytic uremic syndrome (HUS), and central nervous system (CNS) failure (10,17,73). Epidemiological data have demonstrated a high prevalence of E. coli O157:H7 in cattle and their environment but a comparatively low incidence of human infection. This supports the notion that a subset of STEC O157:H7 strains harbored in cattle causes the majority of human disease (18). Genetic heterogeneity among STEC O157:H7 strains has been established using a broad panel of typing m...