Abstract: Objective To understand the main virulent genes and serotypes of non-O157 Shiga toxin-producing Escherichia coli isolates collected in China. Methods The O serogroups were determined by O-antigen biosynthesis gene cluster PCR system combined with the complete O antisera agglutination test. H-antigen types were detected with PCR and sequencing of fliC. stx1, stx2, eae and ehxA genes were screened with PCR. Results Among 434 non-O157 STEC isolates, 82 O serogroups and 28 H types were detected. O20:H30, O2:H32 and O2:H45 were the predominant serotypes. The positive rates of stx1, stx2, stx1+stx2, eae and ehxA genes were 25.35%, 64.98%, 9.68%, 3.92% and 32.95% respectively. Only 15 isolates harbored stx, eae and ehxA genes, which mainly derived from diarrheal patients. Conclusion The serotypes of non-O157 STEC isolates in China are diverse. Detection of eae and ehxA genes could be important for the detection of highly pathogenic strains.