<p><i>Brassica carinata</i> (<i>B. carinata</i>) is a promising non-food biofuel crop with the potential for low carbon intensity biofuel production under suitable management conditions. However, <i>B. carinata</i> requires extensive genetic improvement of many agronomic traits for it to be widely deployed into agriculture systems. Its rapid improvement via gene editing or genetic modification requires robust, genotype-flexible transformation protocols, as current methods are limited and have only been applied to a narrow germplasm base, hindering broader genetic enhancement efforts. In this paper, cotyledonary petioles from five diverse <i>B. carinata</i> genotypes were evaluated for shoot regeneration under different cytokinin treatments. Among the genotypes tested, regeneration efficiency varied substantially across the five <i>B. carinata</i> genotypes. Transient expression of the GUS reporter gene in cotyledonary petioles confirmed high transformation proficiency, with expression frequencies varying from 96.4 to 100% among four genotypes. Consistently, high stable transformation efficiency was achieved for these four genotypes of at least 5%, with the highest efficiency being 12%. Interestingly, transformation and regeneration efficiencies did not correlate, indicating the latter does not serve as a reliable indicator of transformation efficiency when subjected to selection pressures. Transgene integration was validated by PCR and copy number analysis was performed using digital droplet PCR. Furthermore, T₁ seedlings derived from selected transgenic events demonstrated resistance to glufosinate ammonium whereas wild-type seedlings exhibited mortality. Overall, this study presents a significantly improved and reproducible <i>Agrobacterium</i>-mediated transformation method for <i>B.carinata</i>, advancing the potential for rapid genetic improvement and trait development in this promising biofuel crop.</p>