Infections by protozoan parasite Toxoplasma gondii are prevalent worldwide in animals and human. T. gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections to humans and domestic animals. If first be during pregnancy, T. gondii may be transferred vertically by tachyzoites that are passed to the embryo via the placenta. T. gondii may be transmitted horizontally in three phases of the life cycle, ingesting infectious oocysts from the environment or tissue cysts or tachyzoites which are contained in gastrointestinal of many different animals. Transmission may also occur via tachyzoites contained in blood products, texture transplants or non-pasteurized milk. Like rest of the world toxoplasmosis is prevalent in Iran. The present study aimed to determine T. gondii isolates from native cattle in Chaharmahal Va Bakhtiari province located in south west of Iran by molecular methods. In this study, 155 blood samples were collected from native cattle. Genomic DNA was extracted using DNA extraction Kit (Cinna Gen, Iran) according to the manufacturer protocol and PCR was performed using specific primers (ITS-F and ITS-R). Sixteen (6.95%) cattle were positive to T. gondii infection. The positive control samples showed the excepted amplification product specific for T. gondii (171 bp). Although the present results showed relatively low prevalence of T.gondii infection in Chaharmahal Va Bakhtiari native cattle, control and eradication programs seem to be still necessary to prevent the prevalence of this infection factor and economic losses.