Four serotypes of E. coli (O01, 02, 06 and 0128) were used in preparation of a local polylysate oil vaccine that was evaluated in comparison with a licensed imported one for protection of chicken against E. coli infection. Five groups, cach of 20 chickens were vaccinated with the local vaccine subcutaneously (S/C) and intramuscularly (I/M) using single or double vaccinal dose and boostered after 2 weeks. Vaccinated and control chickens were challenged 2 weeks post second vaccination with the four virulent E. coli. strains. No deaths occurred in all vaccinated chickens except one death case in one group. Survivors were necropsied one week post challenge. No gross lesions were observed in all vaccinated groups compared to those of non-vaccinated one. Polylysate vaccine gave similar results to that of the imported vaccine. Anti-E.coli antibodies were measured by an indirect enzyme linked immunosorbent assay (ELISA) in sera of all vaccinated and challenged chickens utilizing the four E. coli vaccinal antigens as individuals and as a polylysate. Moreover, no genetic variation could be detected among the four serotypes after digestion 01 their chromosomal DNA with Xbal enzyme. In conclusion, the polylysate antigens are suitable candidates for vaccine preparation to protect chickens from colibacillosis.