Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx(2) gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx(2) genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx(2) variants and STEC isolates carrying multiple stx(2) genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx(2) sequences with the same PvuII HaeIII HincII AccI type generally cluster together.