The aim of this study was to adjust the basic culture medium for germination of pollen grains for the different species from the genus Hylocereus. Pollen grains from the species Hylocereus polyrhizus Weber and Hylocereus undatus (Haw.) Britton & Rose were withdrawn of anthers from flowers bagged the previous day, before its opening. Then, with a brush, the pollen grains were spread on the surface of Petri dishes containing 20 mL of culture medium. Four experiments were sequentially installed, considering and selecting the best results from the previous experiment to install the next one. Measurements were made on the length of pollen tube under different concentrations of boric acid. Moreover, germination rate readings were performed after 24 h. The pollen viability of species from the genus Hylocereus varies according to species and the pollen germination was influenced by composition and concentration of medium. The culture medium for the germination of pollen grains from H. polyrhizus species must be increased with 100 g L−1 of sucrose, 518 mg L−1 of calcium nitrate and 636 mg L−1 of boric acid, the pH being measured for 5 and the medium solidified with 6 g L−1 of agar. For H. undatus, the medium should be composed of 100 g L−1 of sucrose, 616 mg L−1 of calcium, 619 mg L−1 of boric acid, 6 g L−1 of agar and pH 6. Both species increased the length of pollen tube with the addition of boric acid.