Abstract: The aim of this study was to investigate the distribution of high pathogenicity island (HPI) in Escherichia coli (E. coli) isolated from cows with clinical and subclinical mastitis in China. Milk samples from 750 cows with clinical and subclinical mastitis in seven provinces were used to screen Gram-negative bacteria by MacConkey agar. 115 E. coli isolates were further identified from the Gram-negative bacteria by microbiological tests. Total DNA extraction from E. coli was used to detect HPI genes by PCR. The PCR results showed that in 115 samples, 29, 22 and 20 were positive for genes irp2, fyuA and intB, respectively. Sequence analysis of randomly selected 10 PCR products showed that homology of genes irp2, fyuA and intB were 97.1, 98.2 and 97.2% identical to the published sequences. In addition, HPI+ isolates were associated with fyuA (75.86%) and asn_tRNA locus (68.96%). We further analyzed the association of the prevalence of HPI and O serotypes and found that HPI occurred in several serotypes including O149, O93, O9 and O7. Therefore, we concluded that HPI was widely distributed in the E. coli isolates from cows with clinical and subclinical mastitis in China. The prevalence of HPI is associated with special serotypes in E.coli from cows with clinical and subclinical mastitis.