The mRNA differential display technique was applied to investigate the differences in gene expression in the muscle tissue between black-boned and ordinary (“normal”) sheep. The gene that was differentially expressed was identified through semi-quantitative RT-PCR, and its complete cDNA sequence was then obtained using the rapid amplification of cDNA ends (RACE) method.The nucleotide sequence of the gene was not found homologous with any sheep gene described so far. Sequence prediction analysis revealed that the open reading frame of the gene encodes a protein of 763 amino acids, highly homologous with the hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A (HADHA) of bovine (98%), pig (90%), sumatran orangutan (87%), human (87%), chimpanzee (87%), mouse (83%) and rat (82%) – so that it can be defined as sheep HADHA gene. This novel sheep gene was finally assigned to GeneID: 100192316. Phylogenetic tree analysis revealed that the sheep HADHA gene is genetically closely related to the HADHA gene of cattle. Tissue expression analysis indicated that the sheep HADHA is also differentially expressed In different tissues of black-boned sheep. The present study established the primary foundation for further research on the HADHA gene in sheep.