Ascochyta blight caused by Ascochyta lentis is one of the most important diseases of lentil in the world. This disease has been reported in various areas in Iran causing serious damages every year. Currently, the use of resistant cultivars is considered the most effective and environmentally sustainable strategy for disease control. This study was carried out to determine the genetic diversity of A. lentis populations. For this purpose, samples were collected from eight different regions of Ilam and Kermanshah provinces including: Ilam, Ayvan, Malekshahi, Sirvan, Chardavol, Islamabad, Gilan-e Gharb, Sarpul-e Zahab. After isolation, purification and morphol-ogical identification, genetic variation of 94 A. lentis isolates were assessed using three pairs of SSR primers. Based on molecular analysis, 34 alleles were observed among the populations, and average number of alleles was 1. 688. The amount of marker index (MI) in the ArA03T primers was the highest. The results of AMOVA analysis showed 92% variability within populations and 8% diversity among the populations. Total gene diversity (Ht) and gene diversities between populations (Hs) were estimated 0. 315 and 0. 269, respectively. Gene diversity attributable to differentiate among population (Gst) was 0. 146, while gene flow (Nm) was 2. 903. Cluster analysis based on UPGMA method showed the lowest genetic distance between Ayvan and Sarpol-e Zahab, afterward Ilam and the highest genetic distance between Islamabad and seven remaining populations. This is the first report and comprehe-nsive survey of A. lentis pathogen genetic diversity in Iranian lentil fields. Results of this study can be useful in breeding programs with the aim of producing A. lentis resistant cultivars and developing effective methods for disease control.