Aims: A DNA sequence, from Escherichia coli STEC O145, homologous to O-island 29 from STEC O157 is described, together with a real-time PCR assay for detecting it. Methods and Results: PCR and sequencing were used to identify the 'O-island 29' homologous DNA sequence from STEC O145 (strain VTH34). The sequence divergence between the STEC O145 and O157 'O-island 29' allowed a STEC O145 5'-nuclease PCR assay to be developed. Conclusions: The characterization of a novel locus in STEC O145 has allowed a specific O145 serogroup 5'-nuclease PCR assay to be designed. Significance and Impact of the Study: These findings increase the number of serogroup PCR assays available as alternatives to classical O-serotyping of E. coli .