Heat-resistant molds (HRMs) are common spoilage fungi in heat-processed fruit products, with key species being Paecilomyces variotii and Talaromyces trachyspermus. Their heat resistance is attributed to sexually produced spores called ascospores. As certain culture media favor sexual reproduction and ascospore formation, while others promote vegetative growth, three culture media (PDA, MEA and OA) were tested to determine their ability to support ascospore production. This is crucial for future research concerning the inactivation of these spores. After conducting observations using macroscopic and microscopic techniques to identify the presence of ascospores, our study found that PDA was best for ascospore production in P. variotii (with visible ascospores within 7 days), while MEA was ideal for T. trachyspermus (with ascospores appearing after 14 days).