Walnut anthracnose caused by Ophiognomonia leptostyla is the most important and prevalent fungal disease in most walnut growing areas in Iran. Seventy-five isolates of Ophiognomonia leptostyla, causing walnut anthracnose, were obtained from Juglans regia from various regions of Iran. In order to study the sexual reproduction of the fungus, the isolates collected from various parts of Iran were examined and perithecia were obtained from 11 regions from leaves and in eight isolates from oatmeal agar (OA). These isolates along with five sexually nonfertile isolates were purified as single-ascospore or single-macroconidium cultures. Protoperithecia were readily obtained from leaves collected more than one year back. Perithecia had one beak on leaves and up to four beaks on culture media. As the result 9.3% of isolates were found to be homothallic. In dual cultures of seven non-homothallic, but sexually fertile in vivo, and five sexually non-fertile isolates, no fertile perithecium was produced. Perithecium in homothallic isolates had significantly higher diameter and longer beaks than non-homothallic isolates. There was no significant difference between homothallic and non-homothallic isolates in ascus and ascospore size, colony growth rate and disease index.