In this study we present the genetic analysis of freshly collected garlic landraces (Allium sativum L.) along an eco-geographical transect from northeastern to western Iran covering seven different provinces using ISSR and RAPD markers. A total of 52 polymorphic loci were detected among 31 landrace populations. The percentage of polymorphic bands, the mean effective number of alleles, and the mean gene diversity were 38.82 %, 1.54, and 0.32, respectively. Genetic principal co-ordinate analysis and Structure analysis using 52 polymorphic loci indicated that the germplasm could be divided into two major groups. Principal component analysis (PCA), using geographical and environmental variables suggested the role of both geographical and environmental adaptation in driving and maintaining genetic differentiation between the major groups. In addition, our results showed that the combination of latitude, altitude, and precipitation explains the highest proportion of the variance in the PCA of eco-geographical data. This study shows that geographical and environmental factors together created stronger and more discrete genetic differentiation than isolation by distance alone. These findings emphasize the importance of environmental selection in shaping patterns of genetic structure inferred in Iranian garlic germplasm. We suggest that action should be immediately taken for collecting, protecting and evaluating the genetic diversity of garlic landraces before they disappear in Iran.