Knowledge of the sex of individuals in natural populations greatly facilitates evolutionary ecology, breeding systems and genetics. Therefore, the development of a simple, not stressing and objective sexing test would facilitate conservation of the Short-toed Eagle (Circaetus gallicus), an endangered Accipitridae species living mainly in southern Europe and Asia. A PCR test was used employing primers that amplify two homologous fragments of both the CHD-W gene, unique of females, and the CHD-Z, occurring in the two sexes. The analysis of the PCR products obtained from blood DNA showed a band of about 380 bp, apparently unique in all individuals. The alignment of the sequences of the two fragments revealed that CHD-W is only 9 bp longer than CHD-Z (387 vs. 378 bp) while CHD-Z lacks the restriction site for Asp700I. After digestion male PCR products showed a unique band of 378 bp while fragments belonging to females resolved into three bands (378, 280 and 107 bp). Using feathers as DNA sources, the individual patterns obtained were identical with the corresponding blood DNA samples. This sexing technique is objective and non-invasive and could be useful for verifying the sex ratio theories and improving the management.