Clostridium perfringens (CP) type A and newly created type G strains are the key etiological factors in the induction of necrotic enteritis (NE), an important enteric disease that is responsible for the annual loss of $6 billion in the worldwide poultry industry. Several CP toxin genes were found to be critical in NE pathogenesis in chickens, but limited information is available on the CP lethal toxin tpeL gene. In this study, 19 CP strains isolated from NE-affected chicken farms were characterized microbiologically and molecularly and evaluated for their pathogenicity in commercial broiler chickens. Toxin typing by PCR revealed that all strains tested were positive for the netB toxin gene, but only five strains were positive for the tpeL toxin gene (LLY-TpeL 13, -TpeL 15, -TpeL 17, -TpeL 18, and -TpeL 19, simplified as TpeL 13, TpeL 15, TpeL 17, TpeL 18, and TpeL 19). High levels of TpeL proteins were detected in the concentrated culture supernatant from strains TpeL 13, 15, 17, and 19 by western blotting. Quantitative PCR showed that strains TpeL 13, 15, 17, 18, and 19 harbored a high number of copies of tpeL genes, while TpeL 18 had the highest number of copies of the tpeL gene among all CP strains tested when normalized with copy numbers of 16S rRNA gene as a housekeeping gene marker. The in vivo NE challenge test using multiple oral CP inoculations combined with a high-protein diet showed that TpeL 17 was the most virulent in inducing typical NE lesions, followed by TpeL 19 as the next most virulent, when tested in commercial broiler chickens. Infection with TpeL 17 reduced the growth rate significantly, as shown by reduced relative body weight gain percentage at day 5 postinfection. Availability of the virulent netB⁺tpeL⁺ CP strains is essential for the development of a CP-alone NE challenge model that could provide significant tools for understanding CP pathogenesis and for development of alternative to antibiotics.