The rnpB gene encodes for the RNA subunit of the catalytic ribonuclease RNase P and is present in all bacteria and has both conserved and highly variable sequence regions. Determination of rnpB in 35 Mycobacterium spp. showed species specific sequences for all species except the Mycobacterium tuberculosis complex (four species). High sequence variation was seen in the P3, P15 and P19 regions of suggested secondary structures of the corresponding RNase P RNA molecules. Phylogenetic analysis showed that rnpB gave similar tree topologies as 16S rRNA and hsp65 genes. A combined analysis of the three genes increased the number of nodes with significant support from 10 to 19. The results indicate that rnpB is useful for phylogenetic studies and is a possible target for identification and detection of Mycobacterium spp.