Specific DNA probe has been developed for fast-growing, mycobacterial mutants able to selectively biotransform side chain of plant sterols. The PCR assay, using primers complementary to the sequence of the probe, was shown to distinguish biotechnological mutants from other fast-growing mycobacteria. Moreover, the species identification of biotechnological strains was done using PCR-restriction analysis based on amplification and digestion of the inner part of hsp65 gene (PRA-assay) as well as 16S rRNA sequencing.