Determination of profiles and total contents of betacyanins in cactus fruits of Hylocereus species using chromatographic and spectrophotometric method is described. The investigated species were H. polyrhizus, H. purpusii, H. costaricensis, H. sp. 487 (all red-flesh species and hybrids made among them), and the white- or red-flesh species H. undatus. Hybrids included hybrid 1 (H. undatus white-flesh clone and H. sp. 487), hybrid 35 (H. sp. 487 and H. polyrhizus), and the reciprocal hybrid hybrid 95 (H. polyrhizus and H. sp. 487). Fruits of H. polyrhizus exhibited the highest relative concentration (expressed as percentage of the total HPLC peak area) of hylocerenin, a recently discovered pigment, and a high relative concentration of phyllocactin. Hylocerenin and isohylocerenin, present in fruits at relative concentrations of 11.7 and 5.8%, respectively, are probably responsible for the fluorescent color of the fruit pulp. H. costaricensis fruits have a much higher content of phyllocactin (63.9%), which is almost 4 times higher than the betanin content. These differences in pigment concentrations might explain the differences in red hues of the flesh of these fruits.