Clostridium perfringens (C. perfringens) is a foodborne pathogen that represent a high risk to animals and poultry due to its high outbreak incidences worldwide causing diseases such as necrotic enteritis in chickens, enterotoxemia in rabbits and necro-hemorrhagic enteritis in calves leading to high economic losses in disease prevention and control. This study aimed to investigate the drug resistance pattern and genetic characterization of C. perfringens isolated from different species (chickens, rabbits, and calves) in El-Gharbia governorate, Egypt. The sensitivity test showed that 30 C. perfringens isolates (18 from chickens, 9 from rabbits and 3 from calves) were resistant to erythromycin, spectinomycin and sulphamethoxazoletrimethoprim and sensitive to ampicillin-sulbactam, ofloxacin, cefotaxime, meropenem, cefaclor and amoxicillin-clavulanic acid. Polymerase chain reaction (PCR) revealed that all C. perfringens isolates were type A carrying cpa gene, whereas net B and cpe genes were detected in only (40%) of the 5 tested isolates and were mainly restricted in isolates from chickens. Antibiotic-resistance genes ermB and sul1 were detected in (80%) and (100%) of isolates respectively. The variation of the drug resistance pattern between the current studied locality and others within Egypt may reflect the differences in the levels of the previous misuse of antibiotics. This may be taken as an alarm for the necessity of conducting a whole country well planned and controlled study of C. perfringens antibiotic resistance and their responsible genes to monitor the antibiotics resistance situation allover Egypt and draw a clear map of the current situation and a plan for reducing and even preventing further multidrug resistance (MDR) development of this serious bacteria. C. perfringens net B gene cpa gene chicken