Abstract: Oligonucleotide primers for specific amplification of the hupB gene of Mycobacterium species selected from the group consisting of Seq ID No. 1, Seq ID No. 2, Seq ID No. 3, Seq ID No. 4, Seq ID No. 5 and a method for differentiating Mycobacterium species based on target gene encoding for histone like proteins such as hupB comprising of (a) obtaining DNA from culture or from clinical samples, b) amplifying a part of the target gene encoding for histone like proteins such as hupB of Mycobacterium species using DNA as a template in a polymerise chain reaction with a pair of oligonucleotide primers, c) detecting said amplified fragment of the hupB gene to detect the presence of Mycobacterial species or not and differentiating Mycobacterium tuberculosis from Mycobacterium bovis based on the size of the amplified fragment.