The presence of betacyanin pigments and their isoforms has been detected in the fruit of Hylocereus polyrhizus, a vine cactus native to South America. Along with the known betanin and phyllocactin (6'-O-malonylbetanin), a new betacyanin was structurally elucidated as betanidin 5-O-[6'-O-(3"-hydroxy-3"-methyl-glutaryl)-beta-D-glucopyranoside] (proposed trivial name hylocerenin) by means of electrospray MS/MS, HPLC, and NMR techniques.