Abstract: Objective：To identify the specific types of three clinical isolates of mycobacteria, and analyze their characteristics. Methods：The biochemical features, the sequences of 16S rRNA and rpoB gene, and the antibiotic susceptibility of three isolates were evaluated and analyzed. Results：The specific types could not be identified by traditional biochemical features. However, there were different biochemical features between smooth and rough colonies. The analysis of 16S rRNA sequence showed that there was high homology between the three isolates and the M.chelonei M.abscessus group (similarity>99.7%). Combined with the analysis of ropB gene sequence, the three isolates were identified to be M.massiliense. The antimicrobial susceptibility testing showed that the three isolates were resistant to vancomycin, linezolid and cefoxitin, especially those with the rough colony. Conclusion：M.massiliense is a pathogenic bacterium newly emerged in recent years, and its rough type infection should be paid more attention.