Mycotoxins are secondary metabolites of filamentous fungi, which grow on crops in the field or during storage. They are capable of causing various diseases in humans, thus posing a health risk. PCR-based methods are commonly used for detection of plant pathogens, including filamentous fungi. Here we describe the potential of using real-time PCR for detection of the fungi – possible producers of mycotoxins in grain using primers to the genes involved in mycotoxin biosynthesis. We developed primers based on the sequences of the following genes involved in mycotoxin biosynthesis: ochratoxin A non-ribosomal peptide synthetase gene, involved in ochratoxin A biosynthesis, PSK13 involved in zearalenone biosynthesis, FUM1, involved in fumonisin B1 synthesis, AflR involved in aflatoxin synthesis and isoepoxydon dehydrogenase gene, involved in patulin synthesis. Six samples of wheat grain (harvested in 2019, 2019, 2018, 2016, 2016, 2014), one sample of triticale grain (harvested in 2018), two samples of barley grain (harvested in 2013 and 2014) were analyzed by real-time PCR using those primers and SybrGreen as a dye. Sequence of non-ribosomal peptide synthetase gene, involved in ochratoxin A synthesis, was detected in three out of six analyzed wheat samples, both analyzed barley samples, and triticale sample. PSK1, involved in zearalenone synthesis, was detected in four out of six analyzed wheat samples, one out of two analyzed barley samples, and one triticale sample. FUM1, involved in fumonisin B1 synthesis, was detected in one wheat sample and in triticale sample. AflR involved in aflatoxin synthesis was detected in triticale and one wheat sample. Isoepoxydon dehydrogenase gene, involved in patulin synthesis, was not detected in any of the analyzed samples. Thus, real-time PCR using primers based on the sequences of the genes involved in mycotoxin biosynthesis can be a good tool for early detection of potentially mycotoxigenic fungi in batches of grain and thus for assessment of the risk of mycotoxin formation in food during storage.