<p>Sugarbeet (<i>Beta vulgaris</i> L.), cultivated for its sucrose content, a staple sugar used globally, is affected by several diseases caused by many different pathogens. The application of biotechnological approaches for generating disease-resistance requires a reliable transformation and regeneration system. In this study, we first developed a straightforward regeneration protocol for hypocotyl explants by optimizing various conditions for callus induction to shoot formation. Subsequently, using hypocotyl-derived callus, we established an efficient <i>Agrobacterium</i>-mediated transformation system with the regeneration of whole plants. With the cotyledons, although regeneration is a concern, transient expression of RUBY, a visual marker, was detectable with 32.9% of transformed cotyledons. For the hypocotyl-derived transformed lines 11 out of 22 were molecularly RUBY positive. The frequency of callus induction was higher when the explants were treated with 0.4 optical density <i>Agrobacterium</i> culture, two days co-cultivation and incubation in the dark. The shoot regeneration of 36% was achieved for hypocotyl-derived calli by gradually increasing N⁶-benzylaminopurine, kinetin, and α-napthaleneacetic acid. By optimizing hormone levels, root formation occurred within two weeks. Transgene integration in the regenerated lines was confirmed by sequencing of PCR amplicons. Further, the transgene expression in the lines was confirmed by reverse transcription PCR analysis. The transgenic lines were acclimatized and grown in soil under controlled conditions. The transformation and callus-based regeneration protocols developed in this study can facilitate the production of non-chimeric transgenic lines compared to direct <i>in planta</i> regeneration methods. This way, the first-generation transgenic lines are suitable for evaluating RNAi and gene-editing approaches to target important sugarbeet pathogens, particularly viruses.</p>