Clostridium perfringens (CP) is the etiologic agent of necrotic enteritis (NE) in broiler chickens that is responsible for massive economic losses in the poultry industry in response to voluntary reduction and withdrawal of antibiotic growth promoters. Large variations exist in the CP isolates in inducing intestinal NE lesions. However, limited information is available on CP isolate genetics in inducing NE with other predisposing factors. This study investigated the ability of five CP isolates from different sources to influence NE pathogenesis by using an Eimeria maxima (EM) coinfection NE model: Str.13 (from soil), LLY_N11 (healthy chicken intestine), SM101 (food poisoning), Del1 (netB+tpeL–) and LLY_Tpel17 (netB+tpeL+) for NE-afflicted chickens. The 2-wk-old broiler chickens were preinfected with EM (5 × 103 oocysts) followed by CP infection (around 1 × 109 colony-forming units per chicken). The group of the LLY_Tpel17 isolate with EM coinfection had 25% mortality. No mortality was observed in the groups infected with EM alone, all CP alone, or dual infections of EM/other CP isolates. In this model of EM/CP coinfections, the relative percentages of body weight gain showed statistically significant decreases in all EM/CP groups except the EM/SM101 group when compared with the sham control group. Evident gut lesions were only observed in the three groups of EM/LLY_N11, EM/Del1, and EM/LLY_Tpel17, all of which possessed an essential NE pathogenesis locus in their genomes. Our studies indicate that LLY_Tpel17 is highly pathogenic to induce severe gut lesions and would be a good CP challenge strain for studies investigating pathogenesis and evaluating the protection efficacy for antibiotic alternative approaches.