Powdery mildew caused by Erysiphe necator, is one of the most economically damaging diseases of grapevine throughout the world. This study aimed to identify the mating types of this pathogen in Syria using molecular markers. Three DNA extraction methods: Promega Kit, phenol chloroform isoamyl alcohol (PCI) and SDS were compared. The amount and quality of DNAs obtained by the SDS method were suitable for PCR amplification and other molecular assays. PCR amplification using specific primers (Uncin 144 and Uncin 511) was performed and the expected amplicon of 300-400 bp was obtained from 29 isolates of E. necator collected from different geographical locations and from different grapevine cultivars. Results obtained showed that E. necator may cause atypical symptoms similar to those of downy mildew, in some grapevine cultivars according to the environmental conditions and training system used. Isolates of E. necator were classified into four groups according to geographical locations and grapevine cultivar, based on the observed variation in banding pattern with E07 primer and the constructed phenogram by using UPGMA. RAPD analysis of 39 E. necator isolates using the primer E07 showed the presence of two mating types, and the frequency of each varied depending on the geographical location, with a predominance of the - mating type which did not produce a 1000 bp band using the PCR primer E07.