This study investigated the distribution and characteristics of the Barley yellow dwarf virus (BYDV) species BYDV-PAS, which was recently separated from BYDV-PAV, the most commonly studied BYDV species. Throughout 3 years of experimental monitoring of BYDV incidence, PAS was the most frequently occurring species infecting cereals and grasses in the Czech Republic. Furthermore, Rhopalosiphum maidis and Metopolophium dirhodum were recorded as BYDV-PAS vectors, even though M. dirhodum does not usually transmit BYDV-PAV. In field experiments with barley and wheat, where virus accumulation, symptoms and effect on the yield were tested, BYDV-PAV was more severe than PAS. Infection with the BYDV-PAV isolate resulted in greater expression of symptoms and also in a greater reduction in plant height and grain weight per spike than BYDV-PAS. In a sensitive cultivar of barley (Graciosa), the amount of viral RNA of BYDV-PAV was also significantly higher than that of BYDV-PAS. In a tolerant line (Wbon-123), however, no such differences were found. In conclusion, although BYDV-PAS seems to be dominant in the Czech Republic, BYDV-PAV has the potential to cause more significant crop losses in barley and wheat.