Anti-Müllerian hormone (AMH) acts in maintaining orderly cyclic recruitment of early follicles, suggesting that it is a promising candidate for influencing animal reproductive efficiency. This study aimed to elucidate the effect of a missense mutation of Val566Ile on the structure of AMH protein and the genetic association of Val566Ile and AMH expression with egg production in chickens. Structural perturbations of Val566Ile were predicted by homology modeling. The association of the variant with the number of eggs was tested using a quantitative trait transmission disequilibrium test model. AMH expression in granulosa cells in Lueyang black-boned chickens was compared with that in Nick chickens. The Val566 of AMH is a non-conservative amino acid among mammals and birds, but its hydrophobicity is completely conservative. The substitution of Val566 for Ile566 potentially disrupted hydrogen bonds and solvent accessibility of 22 residues and created a short α-helix in the C terminus of AMH. Despite having striking structure-disrupting potential, the variant was not statistically associated with the number of eggs (P>0.05) in the Lueyang black-boned chickens. We did not detect differential expression of AMH between Lueyang black-boned chickens and Nick chickens (P>0.05). These results confirmed the structural impact of Val566Ile, but suggested that Val566Ile and AMH expression might not be the major genetic determinants for egg production in Lueyang black-boned chickens.