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Late blowing defects in semi-hard and hard cheeses caused by spore-forming clostridia (e.g., Clostridium tyrobutyricum, Clostridium butyricum, Clostridium sporogenes) pose a major issue for the dairy industry. With this study, we applied a multiplex PCR for the rapid and simultaneous detection of clostridia in raw milk for cheese production. Spore detection in milk usually relies on culture-dependent methods, among which the most probable number (MPN) technique is sensitive but time-consuming and nonspecific. We tested two PCR-based protocols: the one entailed direct milk analysis with results obtained within 24 h; the other included an enrichment step and gave results within 72 h. The second protocol was found to be more sensitive; it detected concentrations as low as 100 cells/L for C. sporogenes and C. butyricum and 800 cells/L for C. tyrobutyricum. Both protocols were applied to field samples (211 samples underwent protocol no. 1; 117 samples underwent protocol no. 2) collected from four dairy processing plants in Piedmont. The prevalence of C. butyricum (protocol no. 1: 9.5%; protocol no. 2: 23%) was higher than either C. sporogenes (0%; 9.4%) or C. tyrobutyricum (0%; 6.8%). Protocol no. 2 detected multiple targets in eight samples, indicating that more than one microorganism was present. Our findings underscore the importance of implementing preventive measures and early detection strategies to mitigate the risk of cheese spoilage due to clostridial contamination. [ABSTRACT FROM AUTHOR]