Diseases that are caused by non-tuberculous mycobacteria (NTM) continue to pose difficult clinical problems, and the epidemiological aspect of NTM-caused diseases is of great importance. In the case of Mycobacterium gordonae there is no adequate genotyping scheme. Here we present a potential rapid and reproducible genetic assay that uses trinucleotide repeat sequence-based PCR (TRS-PCR) for genotyping M. gordonae. The proposed method constitutes a useful single-primer PCR screen for genotyping this species. Among 10 TRS-containing primers, after applying (CAC)₄-based PCR to 36 strains of M. gordonae, we found a discriminatory index of 0.975. The accuracy of this analysis was supported by a reasonable reproducibility of 92%. These results were compared with the Enterobacterial Repetitive Intergenic Consensus Sequences (ERIC)-PCR typing scheme which had lower discriminatory index of 0.93 and its reproducibility was only 86.3%.