Rhizomania is one of the most damaging and widely spread diseases in major sugar beet growing regions of the world. The causal agent, beet necrotic yellow vein virus (BNYVV), is transmitted via the fungus Polymyxa betae, which retains it in the field for years. In this study, an RNA silencing mechanism was employed to induce resistance against rhizomania using intron-hairpin RNA (ihpRNA) constructs. These constructs were based on sequences of the BNYVV 5′-untranslated region of RNA-2 or the flanking sequence encoding P21 coat protein, with different lengths and orientations. Both transient and stable transformation methods produced effective resistance against rhizomania correlated with the transgene presence. Among the constructs, those generating ihpRNA structures with small intronic loops produced the highest frequencies of resistant events. The inheritance of transgenes and resistance was confirmed over generations in stably transformed plants.