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Infections caused by non-tuberculous mycobacteria (NTM) often escape detection on traditional diagnostics. We evaluated the ability of heat shock protein 65 (hsp65) polymerase chain reaction and high-resolution melting curve (HRMC) analysis to identify and differentiate Mycobacterium tuberculosis complex (MTC) from NTM in 150 clinical mycobacterial isolates from extra-pulmonary sites. A hundred MTC and 50 NTM isolates were identified. We were able to simultaneously differentiate between seven mycobacterial species using HRMC analysis; a concordance of 100% between hsp65 HRMC analysis, polymerase chain reaction + restriction analysis and hsp65 sequencing was observed.