Necrotic enteritis is an enteric disease of avian species caused by the anaerobic bacterium Clostridium perfringens. The disease is regularly controlled in the broiler chicken industry with antimicrobials in feed but is reemerging in areas such as Europe where there is a ban on antimicrobials as growth promoters. To study prospective therapies, researchers must be able to reproduce this disease in a controlled environment, but this is not always possible because of differences in the pathogenicity of C. perfringens strains. Our objective was to test the potential of five isolates (SNECP43, 44, 47, 49, and 50), taken from field cases of necrotic enteritis, at recreating the disease in a controlled challenge experiment. SNECP43 and 50 were derived from a common clone, with SNECP50 passed in vivo and SNECP43 subcultured in vitro. Four hundred birds were divided into 16 pens, with three pens each receiving one of five treatments, with one control pen. Day-old birds were raised on a high wheat-based diet to promote necrotic enteritis development and were challenged with between 3.4 × 10⁹ and 3.2 × 10¹¹ colony-forming units (cfu) of C. perfringens in feed for a period of 24 hr starting on day 13 of the challenge experiment. Lesion scores were assessed on two birds per pen sacrificed on day 17 and on any dead birds during the 25-day study. Growth performance was assessed up to 25 days, and mortality recorded throughout. Only SNECP50 produced necrotic enteritis mortalities significantly different (P ≤ 0.05) from the control. The five isolates were also typed using pulsed-field gel electrophoresis to assess their genetic relatedness. All epidemiologically unrelated isolates were deemed genetically unrelated, whereas SNECP43 and 50 differed by only a single minor band. Toxin type was assessed using polymerase chain reaction (PCR), which was also used for the detection of the gene encoding the β2-toxin.