One of the most important and elusive diseases of grapevine is Esca disease. Esca is a complex disease that comprises a combination of symptoms caused by various fungi including growth retardation, decrease in productivity and decline of the plant and usually affecting adult plants (8 to 10 years and older). The purpose of this research was to study the genetic variations of fungal agents associated with Esca disease in Khorasan province which was performed by using the molecular marker PAPD. Sampling carried out done from the grapevines of Bojnourd (North Khorasan) and Phaeoacremonium parasiticum, Phaeacremonium aleophilum, Phaeomoniella chlamydospora were isolated from wood brown decay while Fomitiporia mediterranea was isolated from wood white decay. The isolates with the most variability in morphology were selected and DNA polymorphism of the isolates was studied by RAPD-PCR. For this purpose after extracting DNA, RAPD _PCR was performed in 25 microliter reactions with 10 random primers. The results of RAPD analysis demonstrated that remarkable genetic variations existed in F. mediterranea while rather uniformity in Pa. chlamydosporare.. The results showed that RAPD-PCR technique have a great potential in determining genetic variations of the fungi associated with Esca disease. This is the first study of genetic variability in Iranian isolates of fungal agent of Esca using RAPD molecular markers in the populations of this species.