Centaurea wiedemanniana is an endemic plant species native to Turkey. The plant is known to have a narrow distribution in two abutting counties, Bilecik and Eskisehir. Genetic variability within and among 6 known natural populations of this plant was assessed by using RAPD markers. High levels of genetic diversity were detected both at population (PPB = 62.83%, I = 0.301, H-S = 0.196) and species level (PPB = 94.65%, I = 0.429, H-T = 0.278). Analyses of molecular variance showed that among and within-population genetic variation accounted for 71.70% and 28.30% of the total genetic variation, respectively. A high degree of genetic differentiation was also found among populations (G(ST) = 0.223). The genetic structure of C. wiedemanniana has probably been shaped by its breeding characteristics, habitat preference and short distance seed and pollen dispersal. The observed genetic variations suggest that as many populations as possible should be considered in any planned in situ or ex situ conservation programs for this vulnerable endemic species.