We report here the structural basis of CD8 polymorphism in the chicken. Three chicken strains (RPRL Line 7, H.B15.H7, and H.B15. H12) have 14 nucleotide differences in the CD8A cDNA sequence causing eight amino acid replacements in the extracellular part of the molecule. Only two amino acid replacements and four silent mutations were observed in the CD8B cDNA sequence in one (H7) of the strains. Substitutions in CD8alpha were solely responsible for the binding of CD8-specific monoclonal antibodies, as detected by cDNA expression in COS cells. The majority of the amino acid substitutions are located in the immunoglobulin V-like domain and three of the changes (residues 30, 34, and 58) are situated in the putative major histocompatibility complex class I binding CDR1 and CDR2 regions of the chicken CD8alpha. CD8A polymorphism has not been reported in other species and this suggests that CD8A and CD8B have evolved under different selective pressures in the chicken.