Genetic diversity and genome size variability were studied in 16 geographical populations of Linum austriacum (Lineaceae). Genetic diversity parameters were determined in each population based on ISSR molecular markers. AMOVA test, Gst value and Hickory test revealed significant molecular difference among the studied populations. Mantel test showed correlation between genetic distance and geographical distance in these populations. NJ tree and NeighborNet network grouped the studied plant specimens in 3 major clusters. STRUCTURE analysis identified 12 allelic combinations in agreement with K-Means clustering result. These analyses revealed the presence of genetic variability both among and within studied population. The plant specimens of these geographical populations also differed significantly in their genome size.