Some studies have shown that the NetB toxin may be an important virulence factor of Clostridium perfringens associated necrotic enteritis in poultry. Additionally, research has shown that strains of C. perfringens positive for both the netB gene and a second toxin-encoding gene, tpeL, appear to be more virulent than strains with only netB. In the past, detection of these genes has been performed relatively inefficiently using two single locus PCRs. This report describes a novel multiplex PCR developed to detect netB and tpeL simultaneously in C. perfringens strains isolated from cases of necrotic enteritis in broilers, providing a more efficient diagnostic tool in the screening of strains for these genes.