Isatis glauca Aucher ex Boiss. has four subspecies: glauca, galatica YA +/- ldA +/- rA +/- mlA +/-, sivasica (Davis) YA +/- ldA +/- rA +/- mlA +/- and iconia (Boiss.) Davis, naturally growing in Turkey. In this study, 77 accessions from nine I. glauca ssp. populations were characterised in terms of variation in morphological characters, including the number of branches per plant, plant height, diameter of stem base, diameter of middle stem, diameter of apical stem, width and length of fruit, length of fruit stalk, fruit shape, fruit base shape, fruit apices shape, loculus, ondulation, apical process of fruit, fruit weight, basal length of fruit, apical length of fruit, wing width of fruit, loculus length, loculus width, length of seed, dorsal width of seed, lateral width of seed and seed weight. The number of branches had the highest value of coefficient of variation (CV), 0.21-0.86, while the fruit width had the lowest value of CV 0.08-0.20. According to Pearson's correlation and multiple regression analyses, eco-geographical factors had a substantial effect on morphological variation. A principal component analysis explained 90.61 % of morphological variation through the study of five extracted components. A dendrogram, which was constructed according to the between-group linkages method, based on squared Euclidean distances, represented a hierarchical cluster analysis of 77 accessions. The results of the principal coordinate analysis, which showed differentiation according to morphological characters between nine populations, were consistent with the dendrogram. Consequently, morphological variation of I. glauca ssp. and the significant effects of eco-geographical factors on its morphological variation were discussed.