Shiga toxin (stx)-producing Escherichia coli O157 : H7 is a prominent food-borne pathogen. Symptoms in human infections range from asymptomatic to haemorrhagic colitis and haemolytic uraemic syndrome, and there is a need for methods that yield information that can be used to better predict clinical and epidemiological outcomes. IS629 is an insertion sequence notable for its prevalence and variable distribution in the chromosome of E. coli O157 : H7, which has been exploited for subtyping and strain characterization. In particular, IS629 distribution is closely aligned with the major phylogenetic lineages that are known to be distinctive in their genome structure and virulence potential. In the present study, a comprehensive subtyping method in which IS629-typing was combined with stx genotyping was developed using a conventional PCR approach. This method consisted of a set of 32 markers based on the unique distribution of IS629 in the three major phylogenetic lineages of E. coli O157 : H7 and six additional markers to determine the stx genotype, a key virulence signature associated with each lineage. The analysis of IS629 loci variation with the 32 markers allowed us to determine the IS629 distribution profile (IDP), phylogenetic lineage and genetic relatedness of the 31 E. coli O157 : H7 strains examined. An association between IDP typing and stx genotype was observed. The use of both IDP and the stx genotype for strain characterization provided confirmative and complementary data in support of lineage placement of closely related strains. In addition, IS629/stx profiles were in agreement with strain segregation based on LSPA-6 (lineage-specific polymorphism assay) and PFGE subtyping, demonstrating its potential as a subtyping and strain tracking method.