<p>The absence of effective genetic transformation methods in Pitaya species significantly restricts the investigation of gene function and implementation of modern breeding techniques such as gene editing. Establishing an efficient regeneration protocol is a prerequisite for genetic transformation. In this study, a procedure for adventitious shoot regeneration in <i>Hylocereus undatus</i> cv. ‘Cosmic’ is described. Various factors affecting in vitro shoot regeneration from cladode sections were evaluated, including the effects of cytokines, auxins, and light exposure. The best results were achieved on Murashige and Skoog medium supplemented with 4.5&#xa0;µM N6-benzylaminopurine and 0.5&#xa0;µM indole-3-butyric acid, reaching regeneration rates up to 82.1% ± 5.1%. Additionally, the effect of the antibiotic kanamycin was studied. Forty mg L⁻¹ kanamycin completely inhibited shoot regeneration from non-infected explants, whereas 5.4 ± 3.0% of the explants infected with <i>A. tumefaciens</i> harboring the pBINGUSint binary plasmid regenerated at the same kanamycin concentration. Nevertheless, based on GUS assays, none of the regenerated shoots were confirmed as transgenic. The relatively high rate of shoot regeneration achieved is a critical step towards developing effective transformation techniques for this species. The development of such a method will help pitaya breeding progress more quickly by opening the door for functional genomics studies and gene editing.</p>