The objectives of the study were to test diversity among winter barley breeding lines exhibiting various sensitivity to FHB and to find RAPD markers and AFLP markers that will distinguish between susceptible and moderately resistant genotypes. A test of a set of winter barley genotypes artificially infected in field trials by fusaria was carried out. Based on the results from field and laboratory evaluation and deoxynivalenol (DON) content assessment, barley genotypes with different responses to FHB were selected. The genotypes were hybridized and doubled haploid (DH) lines were derived in F1 generation using the in vitro androgenesis method. Initial parental components and derived DH lines were tested for FHB infection and DON content. A set of parental genotypes of winter barley was tested with 80 RAPD markers. Based on analyses of 80 RAPD primers in a set of parental genotypes of winter barley, the primer H15 was selected that provides specific product of 650-bp size for moderately resistant winter barley genotypes. In consecutive detection, this specific product was found in 4 DH lines. During the study, some DH lines were selected that exhibited improved resistance to Fusarium infection. A low infection level and low DON content were found in the winter barley line DH 610 from the combination of Br2611m × Duet. The AFLP technique was used to analyze parental genotypes of winter barley as well as 7 selected DH lines of winter barley. The detected markers can be further evaluated and employed to select breeding materials.