There is increasing incidence of food spoilage and health hazards caused by heat-resistant fungi belonging to the genera Byssochlamys, Thermoascus, and Neosartorya, among others. Their ascospores cannot be sterilized by heating the food. The microbiological risk assessment studies of these fungi during the production of food and beverages indicated that these fungal species or genera in food are associated with different health risks. Therefore, it is necessary to distinguish Byssochlamys, Thermoascus, and Neosartorya from other fungi in the food industry. These genera can be identified by sequence analysis of housekeeping genes such as β-tubulin, but the process is costly and time-consuming. Therefore, rapid and simple PCR-based methods have been developed using specific primer sets for genus- or species-level identification. PCR amplification products are observed to be specific for each of these genera or species and do not cross-react with other fungi associated with food spoilage and environmental contamination. These identification methods are simple, rapid, and highly specific, making them feasible for use in the quality management of food production plants.