Wheat streak mosaic virus (WSMV) has become a re-emerging pathogen in recent years in the Czech Republic. Crop (e.g. wheat, barley, maize) and non-crop grasses from the Poaceae family are the natural hosts of the virus. Here, we report the results from coat protein (CP) gene-sequence analysis of WSMV isolates from wheat crops (four cultivars: Turondot, Bodyček, Avenue, Hymack) and three grass species (Agropyron repens, Phleum pratense, Poa pratensis). Phylogenetic reconstruction of putative CP sequences showed that all tested isolates clustered with existing type B isolates of WSMV (originating from Europe and Asia) rather than type D (originating from USA, Argentina, Australia, and Iran) and type A (originating from Mexico) isolates. Analysis of recombination events showed that Turondot and Hymack isolates recombined with P. pratense, whereas Bodyček and Avenue isolates recombined with a type B isolate (Iran_Saadat-Shahr). The grasses A. repens, P. pratense and P. pratensis share recombination events with type A (Mexico_El Batán), type B (French and German isolates) and type D (Iran_Naghadeh) isolates. The characteristic GCA (Gly276) triplet codon found in type B isolates was conserved in both the wheat and grass isolates. Notably, nucleotide variations were mainly observed at positions nt 381–389, nt 405–460 and nt 486–497 between crop and non-crop hosts. Based on our analysis, we propose that the grass isolates form subtype B1 within the type B isolates of WSMV. Putative CP amino acid sequences in the centre of the protein and in the C-terminal domain (aa 112–260) were significantly more frequently conserved in both wheat and grasses than those in the N-terminal domain (aa 11–80). Collectively, these results indicate that variations exist between crop and non-crop hosts of WSMV.