The present invention relates to a pair of primers specific to mycobacterial species, a polynucleotide of an hsp 65 gene fragment, and a method for the identification of mycobacterial species by using the same. More specifically, the 604-bp hsp 65 gene fragment can be applied to identification methods of mycobacteria such as the comparative sequence analysis method, the probe hybridization method, and PCR-RFLP, which can resolve the problems of a conventional identification method based on bio-chemical characteristics, where the genus mycobacterium covers various species and has a low growth rate, and of the problems of 16s rDNA. Thus, according to the identification method of the present invention, the mycobacterial species can be identified simply, economically, and accurately.