Turnip yellows virus (TuYV; family Solemoviridae, genus Polerovirus) is the most widespread and economically damaging virus of canola (Brassica napus L.) production in Australia. However, no Australian commercial seed companies market TuYV-resistant canola cultivars, and little information is available on the susceptibility of those available. To identify potential sources of TuYV resistance, 100 B. napus accessions from the ERANET ASSYST diversity set were screened in the field and five of these were selected for further phenotyping via aphid inoculation. Furthermore, 43 Australian canola cultivars, six B. napus genotypes with previously reported resistance, and 33 B. oleracea and B. rapa cultivars were phenotyped. All Australian cultivars were susceptible except for 'ATR Stingray'. Stronger resistance to systemic TuYV infection (IR) was identified in diversity set accessions 'Liraspa-A', 'SWU Chinese 3', and 'SWU Chinese 5'. As indicated by lower relative enzyme-linked immunosorbent assay absorbance values (R-E405) in infected plants, resistance to TuYV accumulation (AR) often accompanied IR. Moderate IR was identified in four B. oleracea cultivars and one B. rapa cultivar. Very strong AR was identified in four B. oleracea cultivars and AR of some degree was common across many cultivars of this species tested. The impact of temperature during the inoculation access period or post-inoculation incubation on the resistance identified was examined. Infection rates were significantly higher in resistant B. napus genotypes when inoculated at 16°C than at 26°C, suggesting an increase in aphid transmission efficiency. IR in B. napus genotypes was strong when incubated at 16°C, but weakened at elevated temperatures with almost total breakdown in most genotypes at 30°C. However, infected plants of B. napus and B. oleracea genotypes with AR maintained lower R-E405 values than susceptible controls at all temperatures tested. Novel sources of resistance identified in this study offer potential as breeding material in Australia and abroad.