Anti-Müllerian hormone (AMH), a glycoprotein secreted from the fetal testis, is responsible for regression of the Müllerian duct in the male fetus. The aim of this study was to evaluate the usefulness of serum AMH as a biomarker for diagnosis of cryptorchidism in horses. Serum AMH concentrations were measured in intact stallions, hemi-castrated unilateral cryptorchid stallions, and geldings. In addition, expression of AMH was characterized in cryptorchid testes by immunohistochemistry. Serum AMH was detected in intact stallions (n=11, 13.3 ± 1.8 ng/ml) and in hemi-castrated cryptorchid stallions (n=8, 17.6 ± 3.0 ng/ml), but not in geldings (n=6, all data were below the limit of detection). Immunolabeling for AMH was detected in Sertoli cells of undescended testes from cryptorchid horses as well as those of normal testes. Our findings indicate that the cryptorchid testis after hemi-castration secretes AMH and that serum AMH concentrations may be a useful biomarker for diagnosis of equine cryptorchidism.