Wheat streak mosaic virus (WSMV) has become a re-emerging pathogen in cereal crops in the Czech Republic.  WSMV was first reported in the former Czechoslovakia in the early 1980s, and then no record of the virus was documented until 2009.  The incidence of the virus was recorded in recent years in several winter wheat fields and many grass species.  Here, we surveyed the incidence of WSMV in cereal crops.  The results demonstrated the existence of the virus in winter wheat and volunteer wheat during each year of the monitoring period, which spanned from 2013–2016.  Although the range of infected samples was low (6.4% of the total tested samples), a high incidence of well-distributed virus was recorded.  In at least six fields, the virus reached severe and potentially epidemic levels.  In accordance with our previous report detailing WSMV infection of native grasses, we tested several grass species commonly grown in the Czech Republic.  We found that some grass species acted as experimental hosts and possible reservoirs of the virus; these included Anthoxanthum odoratum (sweet vernal grass), Arrhenatherum elatius (false oat-grass), Lolium multiflorum (Italian rye-grass), Bromus japonicus (Japanese chess), Echinochloa crus-galli (barnyard grass), Holcus lanatus (meadow soft grass) and Holcus mollis (creeping soft grass).  Some of these grass species are also important weeds of cereals, which may be the potential source of WSMV infection in cereal crops.  Several widely used winter wheat cultivars were tested in the field after artificial inoculation with WSMV to evaluate virus titre by RT-qPCR.  Overall, the tested cultivars had a low virus titre, which is associated with mild disease symptoms and may provide a good level of crop resistance to WSMV.