Scald, caused by Rhynchosporium secalis has been the major yield-reducing factor for barley production during the last decade. In this study, pathogenic groups of R. secalis were identified to obtain a global picture of the assembly of isolates involved in Syrian populations which is essential for the development of scald-resistant barley cultivars. To identify a number of pathogenic groups, 49 isolates collected over ten years from major barley growing areas in Syria were evaluated on five differential barley genotypes. Genotypes presented a continuous range of response from highly susceptible to moderately resistant, but none were immune to the disease. A cluster analysis placed isolates in six distinct differential pathogenic groups. Mean disease rating of 39.24% was the separation point between avirulent and virulent reactions. Isolate Rs46 exhibited distinct differential virulence patterns associated with high frequency across all genotypes. Hence, the data presented here provides crucial information for future selection of isolates to develop durable barley scald resistance.