Clostridium perfringens has been implicated as a cause of diarrhea in dogs. The objectives of this study were to compare 2 culture methods and to evaluate a multiplex polymerase chain reaction (PCR) assay to detect C. perfringens toxin genes alpha (α), beta (β ), beta 2 (β2), epsilon (ɛ), iota (ι), and C. perfringens enterotoxin (cpe) from canine isolates. Fecal samples were collected from clinically normal non-diarrheic (ND) dogs, (n = 105) and diarrheic dogs (DD, n = 54). Clostridium perfringens was isolated by directly inoculating stool onto 5% sheep blood agar (SBA) and enrichment in brain-heart infusion (BHI) broth, followed by inoculation onto SBA. Isolates were tested by multiplex PCR for the presence of α, β, β2, ɛ, ι, and cpe genes. C. perfringens was isolated from 84% of ND samples using direct culture and from 87.6% with enrichment (P = 0.79). In the DD group, corresponding isolation rates were 90.7% and 93.8% (P = 0.65). All isolates possessed the α toxin gene. Beta (β), β2, ɛ, ι, and cpe toxin genes were identified in 4.5%, 1.1%, 3.4%, 1.1%, and 14.8% of ND isolates, respectively. In the DD group, β and β2 were identified in 5%, ɛ and ι were not identified, and the cpe gene was identified in 16.9% of isolates. Enrichment with BHI broth did not significantly increase the yield of C. perfringens, but it did increase the time and cost of the procedure. C. perfringens toxin genes were present in equal proportions in both the ND and DD groups (P ≤ 0.15 to 0.6). Within the parameters of this study, culture of C. perfringens and PCR for toxin genes is of limited diagnostic usefulness due to its high prevalence in normal dogs and the lack of apparent difference in the distribution of toxin genes between normal and diarrheic dogs.