The aim of the study was to estimate the utility of the HPLC-based method of mycolic acids analysis to classify Mycobacterium species in routine diagnostic procedure on the basis of own three-year experience. 2142 patients' specimens were examined. 141 AFB were cultured. 36.2% strains were classified as M. tuberculosis complex by HPLC. The identification was confirmed by AMPLICOR MTB (Roche diagnostic, USA). M. xenopi (17.0%), M. kansasii (14.2%) and M. gordonae (14.2%) were the most frequent identified out of nontuberculous mycobacteria. Four mycobacteriosis cases were suspected because of repeated identification of the isolated strains. 136 strains on L-J slant shipped from other centres were identified. We confirm that the HPLC method is highly effective and specific for Mycobacterium species classification, which can be performed in no more than a couple of hours. In our opinion it is a very helpful tool, hard to replace in diagnostic procedure of tuberculosis and mycobacteriosis.