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The objective of the present study was to explore the polymorphism of microsatellite markers localised on chromosomes 3H and 7H in 15 genotypes of barley (Hordeum vulgare L.), spring form (2n = 2x = 14 chromosomes, genome HVHV) from the collection of genetic resources of the Agricultural Research Institute Kroměříž, Ltd. showing various degrees of susceptibility to Rhynchosporium secalis. The selection of SSR markers was based on hitherto achieved knowledge according to which the greatest amount of resistance genes against Rhynchosporium secalis is localised on chromosomes 3H and 7H of barley. We selected 33 SSR markers for the analyses; 17 were localised on chromosome 3H of barley and 16 on chromosome 7H. Out of the total 33 SSR markers, 32 were polymorphous and one mar­ker (Bmac0282) was monomorphic. In total we detected 172 alleles ranging between 101 and 235 bp; the average number of alleles per locus was 5.21. In terms of the polymorphism of the SSR markers localised on chromosomes 3H and 7H the highest polymorphism (60%) was detected in the Bmag0006 and Bmag0021 SSR markers; the lowest in the Bmag0877 and EBmac0713 markers, i.e. 20% and 13.3%, respectively. The average polymorphism based on analyses of 17 SSR markers on chromosome 3H was 37.6% and of 16 SSR markers on chromosome 7H was 31.3%. We also calculated the statistical indicators of the variability rate characteristics of the individual microsatellite markers: diversity index (DI) which ranged between 0.000 and 0.907 (on average 0.704); polymorphous information content (PIC) ranging between 0.000 and 0.906 (on average 0.679); and probability identity (PI) ranging between 0.006 and 1.000 (on average 0.137). On the basis of constructed dendrograms for SSR markers of both chromosomes together it was possible to divide the analysed set into cluster I of genotypes resistant and cluster II of genotypes susceptible and moderately susceptible to Rhynchosporium secalis, and was not possible in dendrograms of individual chromosomes.