The aim of this study was to identify a fungal pathogen that caused necrotic leaf spots in experimental plots of oat in two remote regions of Hungary, the Southern Great Plain and Central Transdanubia. Two monosporic isolates, one from each region, were subjected to morphological and molecular investigations, and their pathogenicity to oat, barley and wheat seedlings was tested by artificial inoculations. Morphology of cultures and conidia matched well the description of genus Drechslera, the asexual stage of Pyrenophora. The natural host and higher pathogenicity of both isolates to oat than to barley and wheat suggested that the fungus represented the primarily oat pathogen P. chaetomioides. Although accurate species identification could not be achieved due to overlapping morphology and host range among the oat and barley pathogenic Pyrenophora spp., PCR amplification and direct sequencing of the ITS1-5.8S-ITS2 region of the nuclear ribosomal DNA revealed 100% identity amongst our isolates and several reference strains of P. chaetomioides, justifying the species identity of the fungus we found on oats. Our study also confirmed an earlier, symptom-based observation about the occurrence of Pyrenophora leaf blotch disease in experimental plots in Hungary, and is the first to prove the presence of its causal agent, P. chaetomioides, based on isolation and accurate species identification.