In　this　study,　reverse　transcription　quantitative　real-time　PCR　(RT-qPCR)　assays　using　species-specific　primers　were　developed　for　the　quantification　of　the　three　major　fungal　species　(Saccharomycopsis　fibuligera,　Rhizopus　oryzae,　and　Monascus　purpureus)　during　the　traditional　brewing　of　Hong　Qu　glutinous　rice　wine.　Species-specific　primers　(Sfi-F/Sfi-R,　Ro-F/Ro-R,　and　Mp-F/Mp-R)　targeting　the　ITS-5.8S　rRNA　gene　and　the　beta-tubulin　gene　sequences　were　designed　and　their　specificity　was　evaluated　by　RT-qPCR　with　the　total　RNA　from　fungal　species　closely　related　to　the　traditional　brewing　of　Hong　Qu　glutinous　rice　wine.　Results　of　RT-qPCR　using　species-specific　primers　showed　100 %　inclusivity　(positive　signal　in　the　presence　of　target　complementary　DNA　(cDNA))　and　100 %　exclusivity　(negative　signals　in　the　presence　of　non-target　template).　The　specificity　of　each　primer　set　was　also　tested　when　the　target　cDNA　was　mixed　with　non-target　cDNA,　and　results　showed　that　there　was　no　significant　difference　(P > 0.05)　in　threshold　cycle　(Ct)　values　obtained　from　pure　target　cDNA　and　target　cDNA　mixed　with　non-target　cDNA.　The　detection　limits　of　the　established　qPCR　assays　were　determined　to　be　101　gene　copies/μL　for　S.　fibuligera　and　102　gene　copies/μL　for　R.　oryzae　and　M.　purpureus,　respectively.　Good　correlations　were　obtained　for　the　tested　species　between　102　and　108　gene　copies/μL　by　qPCR　analysis.　Finally,　the　established　RT-qPCR　assays　were　applied　to　quantify　viable　S.　fibuligera,　R.　oryzae,　and　M.　purpureus　during　the　traditional　brewing　of　Hong　Qu　glutinous　rice　wine.　This　study　standardized　the　RT-qPCR　assays　for　dominant　fungi　associated　with　the　traditional　brewing　of　Hong　Qu　glutinous　rice　wine,　and　it　also　proved　the　usefulness　of　RT-qPCR　assays　for　the　quantification　of　live　cells　in　multi-species　samples.　Results　would　show　great　value　in　scientifically　understanding　of　the　brewing　mechanism　of　Hong　Qu　glutinous　rice　wine.　©　2016,　Springer　Science+Business　Media　New　York.