Objective To evaluate the usefulness of mycolic acid for identification of Mycobacterium species using SMIS. Methods One hundred and eighteen clinical Mycobacterium isolates collected from Beijing Tuberculosis and Thoracic Tumor Research Institute through whole year of 2007 were analyzed. The 118 isolates contain 25 isolates of Mycobacterium tuberculosis and 93 non tuberculosis Mycobacterium identified by PNB method. Mycolic acid analysis using SMIS is evaluated for identification of a broad range of Mycobacteria in comparison with 16S rDNA , 16-23S rDNA ITS sequencing to measure the concordance rate and agreement, and verify the concordance rate and agreement among results of mycolic acid, sequencing and PNB in identifying Mycobacterium tuberculosis and non tuberculosis Mycobacterium. Results The concordance rate between mycolic acid method analysis and DNA sequencing is 92% ( 108/118), of which concordance rate in Mycobacterium tuberculosis complex and non tuberculosis Mycobacterium are 95% (35/37) and 90% (73/81) respectively, agreement of both is great( agreement Kappa value is 0. 96). Through retrospective analysis, the concordance of results between SMIS and PNB method analysis is 90% (106/118)and agreement is well( agreement Kappa value is 0. 73 ), the concordance of results between sequencing and PNB method analysis is also 90% ( 106/118 ) and agreement is well (agreement Kappa value is 0. 74 ),despite the identification results of 11 isolates by PNB method are discordant. Conclusion Mycolic acid analysis by SMIS enables rapid identification of a broad range of clinical Mycobacterium species, which could play an important role in polyphasic identification of Mycobacterium species. Key words: Mycolic acids; Mycobacterium; Chromatography,high pressue liquid