Spot blotch of barley (Hordeum vulgare L.), caused by Bipolaris sorokiniana, is worldwide and economically one of the most important diseases. The structure of the B. sorokiniana population is not uniform. Within isolates of this species, there are strains that differ in virulence and aggressiveness. The aim of the study was to determine the variability of virulence within Polish isolates of B. sorokiniana and to test selected strains of this fungus terms of their pathogenicity in relation to different spring barley cultivars. The diversity of 70 Polish isolates of B. sorokiniana was determined based on the reaction of three spring barley test lines – Bowman, ND5883 and NDB12 with a certain susceptibility to infection by this pathogen and compared to three isolates of B. sorokiniana: ND93-1 classified to pathotype 0, ND85F – pathotype 1 and ND90Pr – pathotype 2. In the population of 70 isolates of B. sorokiniana, two pathotypes – 0 (14 isolates) and 1 (56 isolates) were identified. The mean values of leaf infection index evaluated for lines – Bowman, ND5883 and NDB12 in the case of B. sorokiniana isolates of pathotype 0 ranged: 17.08, 25.42 and 18.13, respectively, and in the case of B. sorokiniana isolates of pathotype 1: 15.57, 59.81 and 17.98, respectively. In the second experiment, the susceptibility of 8 spring barley cultivars to leaf infection by 10 selected isolates of B. sorokiniana (5 of pathotype 0 and 5 of pathotype 1) was tested. The mean value of leaf infection index calculated for analyzed cultivars in experimental combination with pathotype 0 isolates of B. sorokiniana was 1.56, and in the case of isolates of pathotype 1 was 16.58.