Five commercial dairy plants were monitored over a 17-mo period to determine the seasonal occurrence of Clostridium spores in streams from the cheesemaking process. Every 2 mo, samples of raw milk (RM), separated cream (SC), pasteurized and standardized vat milk (PSVM), PSVM + lysozyme (PSVM+L), and manufactured cheese aged for 60 to 90 d were processed for analysis. Molecular diversity of the main species identified was determined using repetitive element palindromic PCR. The mean anaerobic spore counts (μ ± SE) were 3.16 ± 0.054, 3.00 ± 0.054, 2.89 ± 0.059, and 2.03 ± 0.054 log10 most probable number/L for RM, PSVM, PSVM+L, and SC, respectively. Although spore counts did not differ between dairy plants, seasonal variation was observed; spore counts of RM, PSVM, and PSVM+L were higher during winter (June to August) and summer (December to February) months, but no seasonal variation was seen in SC counts. The most frequently isolated species was Clostridium tyrobutyricum, ranging from 50 to 58.3% of isolates from milk and cream samples. Clostridium sporogenes was the second most common species identified (16.7–21.1%); Clostridium beijerinckii and Clostridium butyricum were also found, although at lower prevalence (7.9–13.2%). Analysis of the C. tyrobutyricum and C. sporogenes population structure through repetitive element palindromic PCR indicated a high diversity, with unique isolates found in each positive sample. The occurrence of Clostridia spores in incoming streams to cheesemaking was most prominent in the winter and summer seasons, with higher prevalence of C. tyrobutyricum in the months of June and August.