We focused on the identification of microbes that cause PYF by using DNA fingerprinting techniques, such as pyrosequencing and the development of a qPCR-based test for PYF malt. Tag encoded pyrosequencing of the conserved fungal LSU and ITS regions of rRNA gene were applied to comprehensively assess the fungal population diversity of malts whose PYF status had been determined by a variety of fermentation tests and subsequently identify putative PYF microbes. Fungal taxa were selected to design 16 unique primer pairs for qPCR analysis. After optimization malt samples were analysed by qPCR. The rRNA gene copy numbers of the specific taxa were calculated for each sample. Six primer pairs were of most practical value in determining malt PYF status. These primer pairs were further validated with 200 commercial malt samples. A statistical analysis of qPCR data showed two fungal genera were positively correlated with the occurrence of PYF. The PYF determination was significantly associated with primer-probe pair 1. There is 50% probability of PYF occurrence, if the value of primer-probe pair 1 is at 74+ gene copies (gc)/ng DNA at a 95% confidence interval. The probability of PYF occurrence increases to 90% if the value of primer-probe pair 1 is 1573 gc/ng DNA at a 95% confidence interval. For primer-probe pair 2 the logistic regression on continuous value failed since there was a cut point that equals 74 gc/ng genomic DNA. For primer-probe pair 2 ≤74 gc/ng genomic DNA PYF did not occur while for values >74 PYF always occurred.