A survey of Barley yellow dwarf virus (BYDV) incidence in cereal crops in the Czech Republic over 4 years showed, on average, 13.3% BYDV-positive, randomly tested wheat and barley samples. The cultivated wheat and barley cultivars had different levels of susceptibility to BYDV infection. Field trials were performed with different barley and wheat breeding lines and cultivars, and resistance traits were evaluated after artificial inculcation by the viruliferous aphid vector Rhopalosiphum padi L. with BYDV-PAV. Our results showed high variability of visual symptom score (VSS) and reduction in grain weight per spike (GWS-R) in trials within the tested lines and cultivars. The barley line (WBON 96-123) and cultivars (Wysor, Travira) that contained RYd2 differed significantly from other cultivars in VSS. Line WBON 96-123 and cvv. Wysor and Yatzi showed the lowest GWS-R. Wheat line PSR 3628 and cvv. Altigo, Elan, Sparta, Aladin and Hewit showed significant difference from other cultivars in VSS. PSR 3628, Sparta, and Elan showed the lowest GWS-R. Similar results were obtained from BYDV titre analysis by RT-qPCR corresponding to the VSS and GWS-R traits. A low virus titre corresponded to low VSS and GWS-R. Hence, our results suggest that laborious and time-consuming GWS-R analysis could be replaced in some cases by qPCR-based BYDV titre analysis and, together with VSS evaluation, could enhance the efficiency of resistance assessment.