A set of 42 winter wheat cultivars developed from nine populations were evaluated for distinctness. Three marker systems: morphological markers, gliadin allele profiles and microsatellites were used to analyze distinctness between sister cultivars. The morphological based distinctness tests for wheat are based on a crop-specific set of characters that comply with UPOV guidelines. The morphological markers were quite informative although they were not capable to estimate the distances/distinctness between two pair of sister cultivars Evropa 90 and Evropa and Novosadska rana 3 and Novosadska rana 2. The gliadins allele profiles were the least efficient to estimate distinctness between sister line cultivars. This system was not capable to distinguish even cultivars developed from different populations. Sister line wheat cultivars were fingerprinted with 19 wheat microsatellites markers. A total of 106 alleles were detected at 19 wheat microsatellite loci, resulting in an average allele number per marker of 5.6. The number of markers was sufficient to distinguish among most sister line cultivars. Only one pair of sister cultivars, Lozničanka and Kosovka, derived from the same cross could not be distinguished. Comparing all marker systems to evaluate distinctness of sister line wheat cultivars the most efficient was microsatellite markers while gliadin allele profiles was the least efficient. Correlations between matrices based on pedigree data and morphological marker, gliadin profiles and microsatellites were significant but not large. [Projekat Ministarstva nauke Republike Srbije, br. 31066]