The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.