We retrospectively investigated NTM musculoskeletal infections to understand the clinical characteristics as well as the diagnostic performance of molecular tests as a useful supplementary test. A retrospective cohort study was conducted from January 2016 to December 2019. Intraoperative specimens for culture and pathological analysis were obtained during surgery under sterile conditions. AdvanSure TB/NTM real-time PCR (LG Life Sciences, Seoul, Korea) and PCR-reverse blot hybridization assay (REBA, REBA Myco-ID; YD Diagnostics, Yongin, Republic of Korea) was performed directly from tissue specimens for the rapid and accurate identification of 20 mycobacterial species. A series of 24 patients was identified during the study period. Agricultural/aquatic exposure were predisposing factors in 9 patients, trauma in 4 patients, but no predisposing host factor was identified for 11 patients. Twenty-two patients presented with tenosynovitis and two with arthritis with substantial delays between initial presentation of symptoms and microbiologic diagnosis, with a median of 147days (range 5-396days). Pathologic tissue examination of musculoskeletal NTM infections was positive for chronic granulomatous inflammation in 18 cases. Fifteen were positive in the culture study, and nine were positive only in the molecular study using tissue specimens. All 6M. ulcerans/M. marinum isolates were identified only by PCR. This study is an important reminder that NTM infections should be a part of the differential diagnosis in patients with chronic musculoskeletal infections refractory to regular treatment. Although molecular test cannot replace the conventional smear and culture methods, it can be used as a useful supplementary test especially in diagnosing M. marinum infection.