Late blowing, a microbiological spoilage in hard and semi-hard cheese caused by Clostridium spores in raw milk, results in high economic losses for cheese producers. This study compared the sensitivity of the newly developed multiplex qPCR method which employing novel oligonucleotide primers and fluorescent TaqMan probes, and the culture-based most probable number (MPN) method in detecting the late blowing agent Clostridium species in traditional Turkish cheese. A total of 50 naturally contaminated cheese samples obtained from producers were analysed by both methods. Clostridium tyrobutyricum was the most common species occurring in 74% of the cheese samples, followed by C. butyricum and C. sporogenes occurring in 50% and 16% of the samples, respectively. The results of the two methods were consistent in 42 out of the 50 (84%) cheese samples. Our results indicate that the multiplex qPCR method is more sensitive than the MPN method. The multiplex qPCR method provided a favourable alternative to traditional cultural methods. This alternative molecular method has great potential in the laboratory and in the field for the rapid detection of late blowing of cheese samples.