Colibacillosis, an infectious disease of chickens, is caused by avian pathogenic Escherichia coli (APEC); however, in addition to APEC, other pathogens are also frequently isolated from chickens affected with colibacillosis. Therefore, experimental infections in chickens are necessary to evaluate the pathogenicity of APEC isolates. Recent studies have shown that embryo lethality assays can be used as an alternative method to evaluate the pathogenicity of E. coli. In this study, to determine the important virulence genes associated with the pathogenicity of E. coli, 67 strains of E. coli that possessed different combinations of eight representative virulence genes (cva/cvi, vat, tsh, iucD, papC, irp2, iss, and astA) were isolated from broilers with colibacillosis in Japan, and the chicken embryo lethal assay was conducted. The genes vat, papC, and irp2 showed strong correlation with the level of virulence in E. coli. Our study provides useful information about the important virulence-associated genes in relation to the pathogenicity of E. coli in Japanese chickens.