HPLC is the most useful method to analyze various species of mycobacteria by using mycolic acids. The purpose was to prepare a library containing chromatographic patterns of mycolic acids derived from reference species Mycobacterium, which had been cultivated in standard conditions. 28 reference strains (27 ones from American Type Culture Collection and one cultivated from the vaccine M. bovis BCG) were used. The analysis of mycolic acids involved chromatographic separation of their bromophenacyl derivatives according to Centers for Disease Control recommendation. Mycolic acids profiles formed by HLPC were reproducible for all reference species in this study. Standard deviation of relative retention time of every peak did not exceed 2.5%. The species included into M. tuberculosis complex beyond M. bovis BCG shared the same mycolic acids pattern. HPLC is the only mean to distinguish M. tuberculosis from M. bovis BCG. The other studied stains had species specific patterns which differed from M. tuberculosis complex and M. bovis BCG. The prepared library comprising 28 reference elution profiles of mycolic acids from known mycobacteria species can be applied in diagnostic procedure of tuberculosis and mycobacteriosis.