To study the existence of Clostridium septicum as probable agent associated with currently occurrence of black quarter in Urmia area, sheep-dung samples were collected. The bacteriological examinations on suspicious gram positive anaerobic spore-bearing bacilli which are in Clostridia genus include isolation and biochemical identification were performed. The results showed 28 different types of Clostridia contain of Cl. Septicum (6 isolates), Cl. Perfringens (5 isolates), Cl. novyi (4 isolates), Cl. fallax (4 isolates), Cl. innoccum (3 isolates), Cl. subterminale (2 isolates), Cl. carnis (2 isolates), Cl. ramosum (1 isolate), Cl. bifformentanse (1 isolate). All six isolated of Cl.septicum were confirmed by indirect florescent antibody test while other species were negative. PCR was performed for confirmation of isolation by using designed primers with respect to a segment of hemolysin gene (alpha or lethal toxin). All six isolates showed 270-bp band on agarose gel electrophoresis, suggesting conserved sequence for lethal toxin in Cl. septicum isolates. In this work we have isolated different Cl.septicum from sheep-dung in Urmia and biochemical, indirect immunoflorescent antibody and polymerase chain reaction tests suggesting that PCR could be selected as specific and quick method for identification of this bacterium.