Characterization of species-specific molecular markers and development of a method for identification of Indian deer species is necessary to monitor illegal trade of parts and products for better conservation and management of the endangered species. In this investigation, we characterized the 12S rRNA gene sequence for differentiation of Indian deer species and developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method for their identification. Universal primers were used for the amplification of the mitochondrial 12S rRNA gene from genomic DNA of chital or spotted deer, hog deer, barking deer, sika deer, musk deer and sambar. PCR products of chital, hog deer and Himalayan musk deer were cloned and sequenced for the first time. Among the Indian deer species, more than 90% similarity was observed in the mitochondrial 12S rRNA gene. The sequences of the above deer species were restriction mapped with the help of Lasergene (DNAstar Inc., Madison, WI, USA). PCR amplicon of these deer species were subjected to restriction digestion with Rsa1, Dde1, Bsr1 and BstSF1 endonucleases that showed a species-specific RFLP pattern. This technique provides a reliable and efficient tool for identification of deer species using a variety of biomaterials.