Purpose: Clostridium perfringens is an important pathogen in both human and veterinary medicine. Necrotic enteritis (NE) is the most clinically dramatic bacterial enteric disease of poultry induced by C. perfringens and is more prevalent type of clostridia genus isolated from the intestinal tract of ostrich (Struthio camelus). The pathogenicity of this bacterium is associated with the production of extracellular toxins produced by some of its strains, such as NetB and TpeL toxins. The exact role of these toxins in NE pathogenesis is still controversial. In the present study, C. perfringens isolates from healthy and diseased ostrich flocks analyzed to determine the presence of NetB and TpeL toxin genes (netB and tpeL). Methods: Thirty six isolates of C. perfringens were obtained from NEpositive and NE-negative ostrich flocks in Khorasan-e-Razavi province in Iran and analyzed by two separate single PCR assay to determine the presence of netB and tpeL genes. All isolates used in this study genotyping by multiplex PCR and confirmed as type A and C. Results: Results showed that the netB gene was present in 8 (50%) of the 16 isolates obtained from NE outbreaks and were not present in any isolate from healthy ones. The tpeL gene was present in 6 necrotic enteritis isolates (37.5%) and 2 healthy ostrich flock isolates (10%). Conclusion: Statistical analysis using Fisher exact test indicate that there is no statistically significant difference between healthy and diseased flocks for tpeL (p=0.103) but for netB was (p<0.001). These results suggest that tpeL is not a necessary virulence factor for the development of necrotic enteritis for many strains of C. perfringens. More research is necessary to determine the prevalence of this gene on a global scale, which would indicate its importance as a virulence factor to the C. perfringens population as a whole

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