Objective:To establish a real-time fluorescence-based PCR method for the rapid identification of mycobacteria in skin biopsies.Methods:Primers and probes targeting the genus Mycobacterium and 14 common Mycobacterium species were self-designed, and a real-time fluorescence-based PCR detection system/method was established. Twelve standard strains of Mycobacterium were used for in vitro validation. Tissue samples were collected from 119 patients clinically diagnosed with cutaneous mycobacterial infections at the Department of Dermatology and Venereology, Peking University First Hospital from 2019 to 2021, and were tested with the established real-time fluorescence-based PCR method. Results:In vitro testing demonstrated that the method had good sensitivity and specificity for all the 12 standard strains of Mycobacterium. Among the 119 skin biopsy samples from patients clinically diagnosed with cutaneous mycobacterial infections, the culture positivity rate was only 21.0% (25/119) ; the real-time fluorescence-based PCR yielded a positivity rate of 76.5% (91/119) for the detection of mycobacteria, and 44.5% (53/119) were identified to the species level with Mycobacterium marinum as the predominant species; 31.9% (38/119) were identified only to the genus level. Conclusion:A real-time fluorescence-based PCR method has been established for the efficient detection of mycobacteria in skin tissue samples; however, there remains a possibility of missed diagnoses, other molecular diagnostic techniques are required to be combined to improve the sensitivity and specificity.