Recently, the use of the dry yeast of Zygosaccharomyces roaxii M2 for miso (soybean paste) fermentation has been established. A molecular monitoring method was developed and validated in this study to analyze the population of Z rouxii M2 during the fermentation. The method was based on the restriction patterns of internal transcribed spacer (ITS) regions of the rDNA using HaeIII and Hhal. Among the homologous ITS regions of Z roaxii strains, Z rouxii M2 produced diagnostic bands by which it can be differentiated from the other strains used. The specific restriction bands were due to the difference in nucleotide sequence of two different copies of ITS of Z. rouxii M2. Both ITS copies showed 94% sequence similarity but a 13-bp nucleotide substitution and a 19-bp deletion were found in the ITS1 region. Phylogenetic trees were constructed based on ITS and 18S rDNA sequences and it was found that the ITS sequences provide better resolution for the classification of Z rouxii M2. Since Z rouxii M2 is a promising strain for use in miso fermentation as a dry starter, the method developed is significant in terms of industrial application in monitoring the growth of Z rouxii M2 in miso fermentation.