Highly pathogenic H5Nx avian influenza viruses constantly threaten the poultry industry and humans and have pandemic potential. These viruses continuously evolve, requiring a universal vaccine to protect chickens from members of diverse clades. The purpose of this study was to develop an H5 cleavage-site peptide vaccine containing polybasic amino acids (RRRK) to completely protect chickens from H5N6, H5N8, and H5N1 avian influenza viruses. Chickens were immunized with various doses of a keyhole limpet hemocyanin (KLH)-conjugated H5 cleavage-site peptide vaccine containing RRRK. The effect of RRRK was evaluated by comparing the survival rates of chickens immunized with vaccines either containing or lacking RRRK. The ability of the RRRK-containing vaccine to confer long-term protective immunity was also assessed. We found that protection was dependent on the number of antigens in the vaccine containing RRRK. Chickens immunized intramuscularly with two doses of 5 μg of the vaccine containing RRRK were completely protected, but those immunized with fewer than two doses of 3 or 1 μg were not protected. Chickens immunized with the vaccine lacking RRRK were not protected, suggesting the importance of the polybasic amino acids in conferring immunity. Our results suggest that conserved H5 cleavage-site peptides with polybasic amino acids may be a potential universal vaccine to protect chickens from various emerging clades of H5Nx avian influenza viruses.