The practice of animal sacrifice was widespread in ancient societies, and the study of sacrificial animals holds significant importance. In this study, three samples excavated from the Royal Sacrificial Site of the Northern Wei Dynasty in Inner Mongolia Autonomous Region, China were initially identified as Cervinae subfamily animal based on their morphology. Through alignment analysis and principal component analysis, these samples were identified as belonging to the species Cervus canadensis. Meanwhile, through phylogenetic analyses and genetic distance calculations, it was determined that all three samples were of the Cervus canadensis alashanicus subspecies, proving the utility of ancient DNA technology in accurate subspecies identification. The analysis of the mitochondrial D-loop region revealed that the ancient Cervus canadensis alashanicus dated to 3800 BP and 5100 BP differed significantly from the modern ones in opposition to 1500 BP samples which were similar to them. The low genetic diversity of modern Cervus canadensis alashanicus was formed at least 1500 years ago and have lasted till today. In addition, as suggested by historical documents and archaeological studies, it is evident that the reverence for deer within the Tuoba Xianbei population had become a significant element of their culture and belief system. The identification of Cervus canadensis species in this study serves as evidence of the Northern Wei Emperor’s utilization of totem animals as sacrificial offerings to heaven.