Zymoseptoria tritici is a fungal pathogen causing losses in wheat yields. Here, we present new primer sets for species-specific identification of this microorganism in wheat leaf samples using conventional PCR. Primer sets were validated in silico using tools available in genetic databases. Furthermore, in vitro tests were also carried out on 190 common wheat samples with visual symptoms of Septoria tritici blotch (STB) collected in Poland in three growing seasons (2015, 2016, 2017). The designed primer sets showed full hybridization to the available genetic resources deposited in the NCBI GenBank database, and their high specificity and sensitivity were demonstrated on wheat leaf samples and selected fungal strains.