BACKGROUND: Clostridium perfringens is an important animal pathogen that causes severe loses to the livestock and poultry industries. Therefore, bacterial detection is believed to be of particular importance. OBJECTIVES: The present study aimed to identify Iranian isolates using conventional and molecular methods and to evaluate their toxicity. METHODS: In this work, 23 Clostridium perfringens type B isolates were examined via microbiological and biochemical tests. Subsequently, they were subjected to PCR technique for the final confirmation. After culturing of the isolates in specific medium, the minimum lethal dose test was performed. The most toxigenic isolate and reference strain was prepared the enterotoxaemia anaculture vaccine. Serum neutralization test was performed on the experimental inactive vaccines. RESULTS: The results revealed that etx and cpb gene could be found in all of the isolates, yet cpb2 gene was found in 65.2 % of the isolates. The minimum lethal dose ranges for these bacteria was less than 1/10 to more than 1/900. The results of serum neutralization in Iranian isolate and reference strains were 5 and 10 IU / ml, respectively. CONCLUSIONS: The findings herein implied that strain 1795 with high toxicity could be used in vaccine production. Of course, for use in production, further research on target animals is needed.