Turnip yellows virus (TuYV; family Solemoviridae, genus Polerovirus, species Turnip yellows virus) is a genetically diverse virus that infects a broad range of plant species across the world. Due to its global economic signi cance, most attention has been given to the impact of TuYV on canola (syn. oilseed rape; Brassica napus). In Australia, a major canola exporting country, TuYV isolates are highly diverse with most variation concentrated in open reading frame 5 (ORF 5) which encodes the readthrough domain (P5) component of the readthrough protein (P3P5) which plays an important role in host adaptation and aphid transmission. When analysing ORF 5, Australian TuYV isolates form three phylogenetic groups with just 45 to 49% aa identity; variants P5-I, P5-II and P5-III. Despite the possible implications for TuYV epidemiology and management, research examining phenotypic differences between TuYV variants is scarce. This study was designed to test the hypothesis that three TuYV isolates, representing each of the Australian P5 variants, differ phenotypically. In particular, the host range, vector species, transmissibility, and virulence of isolates 5414 (P5-I 5414 ), 5509 (P5-II 5509 ) and 5594 (P5-III 5594 ) were examined in a series of glasshouse experiments. Only P5-I 5414 readily infected faba bean (Vicia faba), only P5-II 5509 infected chickpea (Cicer arietinum), and only P5-I 5414 and P5-III 5594 infected lettuce (Lactuca sativa). Myzus persicae transmitted each isolate, but Brevicoryne brassicae and Lipaphis pseudobrassicae did not. When using individual M. persicae to inoculate canola seedlings, P5-I 5414 had signi cantly higher transmission rates (82%) than P5-II 5509 (62%) and P5-III 5594 (59%). As indicated by enzyme-linked immunosorbent assay absorbance values, P5-I 5414 reached higher virus titers in canola than P5-II 5509 which, in turn, reached higher titers than P5-III 5594 . P5-I 5414 was also more virulent in canola than P5-II 5509 and P5-III 5594 , inducing more severe foliar symptoms, stunting and, in one of two experiments, seed yield loss.Results from this study compared to those of previous studies suggest that analysis of ORF 5 alone is insu cient to assign isolates to coherent strain categories, and further sequencing and phenotyping of eld isolates is required.