The present study was undertaken to standardize PCR assay for detection of C. perfringens, alpha toxin and enterotoxin from seafood samples collected from selected dams, freshwater lakes, ponds, retail shops and local markets of Telangana State, India and compare with conventional cultural methods. The specificity for C. perfringens, alpha toxin and enterotoxin were tested using primers from 16S rRNA, cpa and cpe genes with 3 Clostridium spp. and 7 other organisms which gave a specific 481, 324 and 233 bp products for C. perfringens, alpha toxin and enterotoxin respectively. The minimum detection level with pure C. perfringens culture was found to be 2.5 CFU/ml. Fluid Thioglycollate broth was superior than Robertson Cooked Meat Broth and 24h incubation was better than 18h. Screening of 300 seafood samples (100 each of fish, crabs and shrimps) revealed that 44 (fish-16, crabs-12, shrimps-16) and 64 (fish-24, crabs-16, shrimps-24) were positive for C. perfringens by cultural and PCR methods, respectively. Out of 64 sea food samples positive for C. perfringens by PCR, 8 (fish-4, crabs-2, shrimps-2) were positive for cpe gene and all PCR positive samples, alpha toxin gene was present. The mean viable counts (CFU/g) are 110, 98 and 102 in fish, crabs and shrimps, respectively.