[Objects] To analyze the molecular epidemiology of Mycobacterium gordonae strains from patients and environments in the hospital. [Subjects] A total of 46 clinical strains were obtained from patients registered at the NHO Kinki-chuo Chest Medical Center and 3 strains from hospital environments. [Methods] By using genetic data from the 16S rRNA gene and hsp65PRA, pulsed-field gel electrophoresis (PFGE) assessment of their intraspecies variability and epidemiology was carried out. [Results] Strains from six patients and environmental cultures exhibited the different genotypes of 16S rRNA gene sequencing and the hsp65PRA type. The PFGE analysis suggested no pseudo-outbreak and showed a polyclonal infection in one patient. [Conclusion] These findings suggest that we should maintain effective surveillance of environments in the hospital and continuously perform molecular epidemiological investigations for infection control of M.gordonae.