The aims of this study were to determine if the chicken embryo lethality assay and the presence of 9 virulence-associated genes of Escherichia coli were correlated and to discover which virulence genes contributed most to embryo lethality. We examined 58 E. coli strains isolated from visceral organs of chickens with colibacillosis for the presence of 9 virulence genes (fimC, tsh, fyuA, irp2, iucD, cvi/cva, iss, astA, and vat) by PCR. The gene FimC (type I fimbriae) was detected with the highest prevalence in 93.1% of the isolates, followed by iucD (67.24%), iss (58.62%), tsh (34.48%), cvi/cva (34.48%), fyuA (32.76%), astA (31.0%), irp2 (27.59%), and vat (17.24%). The embryo mortality ranged from 5 to 100%; however, most of the isolates were moderately or highly virulent. High positive correlations were observed between the presence of virulence genes and chicken embryo lethality. In addition, presence of the iucD (aerobactin) gene was the trait that best contributed to embryo mortality by using the multivariate model. These results suggest that expression frequency of these 9 virulence genes is associated with embryo mortality, and the gene that best predicted embryo mortality was iucD.