[Objective] In order to improve the detection efficiency of high-pathogenicity island (HPI) in avian Escherichia coli isolates, investigate the homology of its int genes and irp2 genes, and finally reveal the transfer regularity of HPI in avian E. coli. [Methods] Orthogonal experimental design was used to optimize multiple-PCR amplification system for irp2 and fyuA, which belong to the core genes of HPI, and avian E. coli clinical isolates were detected with this method. The genetic homologies of irp2 and int genes amplified from seven detected HPI-positive E. coli strains were analyzed. The distribution characteristic of int was analyzed comparing with the ERIC-PCR result of the seven HPI-positive E. coli strains. [Results] The core genes of HPI were specifically amplified by the multiple-PCR method. ERIC-PCR analysis shows that the degree of difference between the HPI-positive strains was higher than 5%. A great conservation (higher than 99% homology) of irp2 genes in HPI-positive E. coli strains was found, and all of the int genes were located in the vicinity of asn-tRNA, but not all of them were highly conserved. [Conclusion] We have established a multiple-PCR method which can be applied to the laboratory diagnosis test and epidemiological investigation of HPI, and suppose that the way of HPI horizontal transfer may be mainly based on the homologous recombination in the HPI-adjacent sequences.