Chickpea (Cicer arietinum L.) is one of the most important crops in the world. After bean and pea, chickpea is the most important cold season legume. Weeds are one of the most important threats to chickpea production worldwide. Due to the sensitivity of chickpea to herbicides, the majority of herbicides are used pre-emergence and the use of post-emergence herbicides is limited, and therefore weeds cause a significant decrease in chickpea yield. Therefore, herbicide-tolerant chickpea cultivars that have a higher flexibility for post-emergence herbicide application are needed to improve the chickpea yield. In this study, using seed bioassay and PCR method, resistance mechanism of Iranian chickpea cultivars to Pursuit herbicide was investigated. The results showed a significant genotypic and phenotypic variation among Iranian chickpea cultivars for tolerance to the Pursuit herbicide. The results did not show a difference between the target genes of Pursuit herbicide, ALS1 and ALS2, in all investigated cultivars with that of the reference sequences in the GenBank. This proves that the resistance observed in different chickpea cultivars to the herbicide Pursuit is not associated with the target site resistance mechanism and probably follows a non-target resistance mechanism. The superior genotypes of this study (Bivanij, Aksou, Mansour, TDS-Maragheh90-400 and TDS-Maragheh90-358) can be recommended to farmers and also suggested as parents to produce natural herbicide resistant chickpea plants in breeding programs.