Apple scab [ Venturia inaequalis (Cke.) Wint.] is the most serious disease of apple worldwide which causes an extensive economic losses due to the negative impact on quality and quantity of apple production. Almost all cultivated apples in Iran are susceptible to V. inaequalis and fungicides application is routine to limit the outbreak of the disease. Overuse of fungicides treatment, causes environmental and economic concerns and also contributed to merge new resistant strains of the pathogen. Therefore, it is necessary to direct all efforts toward the breeding of scab-resistant apple cultivars. The genetic homogeneity of both the cultivated apples and the associated V. inaequalis population in the regions away from the center of diversity has led to assume that, the wild Malus species of East Asia remaining as a substantial resource for apple scab resistance. Despite the existence of various apple genotypes in Iran, the presence of apple scab resistance genes has not been studied yet. Besides, to develop resistant cultivars, it is necessary to understand virulence structure of V. inaequalis and the evolutionary forces that shaping its pathogenesis. Hence, considering the exposure of new races for overcoming scab resistance genes and their prevalence, studying of genetic variation among Iranian isolates of V. inaequalis is more important. In this study the genetic structure of 45 isolates of V. inaequalis was evaluated by microsatellite marker. To do so, the leaves and fruits of apples distorted from scab infection were collected from different apple orchards in Iran during summer 2013. The specimens were cultured on MEA media containing apple extract with 50 ppm from each of Chloramphenicol and Rifampicin antibiotics. DNA was extracted from both cultured mycelia on MEA and also directly from leaves using Chelex ® 100. The isolated V. inaequalis were identified molecularly by ITS5/ ITS4 primers and genetic structure among and within fungal populations were investigated using 12 SSR primer pairs in PCR reactions. After running the PCR products on 6% denaturing sequencing gel, bands were scored and the amplicons were analyzed using PowerMarker V 3.25 software. The dendrograms were drawn based on Nei 1983 and Neighbor Joining method. AMOVA Analysis was performed by Arlequin V 3.1 software. The results demonstrated that the number of amplified alleles in fungi locus varied between 2 to 21 with the average of 10.33 alleles per locus. The average of gene diversity and PIC value was 0.7995 and 0.7739 respectively. The SSR marker was highly polymorphic and could separate the isolates. The populations were divided into four sub-clusters according to the geographical distribution. The dendrogram and percentage of variation among the populations (23.74%) suggest that the V. inaequalis populations distributed homogeneously throughout different regions in Iran because of gene flow, while variation within population was very high due to annual sexual reproduction and several asexual cycles per season. Consequently, the isolates were divided into 19 clusters with percentage variation of 76.36%. The index (0.2374) showed high diversity among the subpopulations of pathogen. The close emplacement of isolates of V. inaequalis from the same varieties of apples in one cluster indicated their co-evolution with the host. The high genetic diversity of V. inaequalis and long lasting apple cultivation in East and West Azerbaijan and Ardabil provinces and also proximity of Khorasan Razavi province to apple origin regions, raise the notion that the isolates of V. inaequalis have been originated from the northern part of Iran. To detect apple scab resistance genes in 28 domestic cultivars