The polymorphism of the short fragment of the heat shock protein 65 encoding gene was evaluated by the PCR - RFLP technique described by Telenti and further developed by Devallois for identification of mycobacterial species in routine laboratory work. We analysed 58 strains representing 25 different mycobacterial species (24 reference strains and 34 clinical isolates). The results obtained by PCR-RFLP and HPLC identification techniques were highly concordant The results were compatible for 87,5% (21 / 24) reference strains and for 97,1% (33/34) clinical isolates. The PCR - RFLP method allowed for accurate identification mycobacterial species, especially pathogenic strains. Restriction patterns obtained for 25 species of Mycobacteriaceae genus could help in constructing the data base and algorithms used in routine laboratory practice.