We investigated physiological differences in watermelon [Citrullus lanatus (Thunb.) Matsum. et Nakai] fruits among seeded diploid and seedless triploid fruits, N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU)–treated seedless fruits, and soft-X–irradiated pollen-pollinated seedless fruits to investigate the effect of the presence or absence of seeds on water relations and sugar content. We picked fruits at 20 and 40 days after anthesis and sampled flesh at the center, around the seeds, and near the pericarp to measure water status and sugar content. There were no significant differences between seeded and seedless cultivars in sugar contents or in water and osmotic potentials of the flesh, although the latter two were decreased at 40 days. CPPU and soft-X–irradiated pollen eliminated mature seeds, but there were again no significant differences in sugar contents or water status between seeded and seedless fruits. Thus, the presence or absence of seeds did not influence the sugar content or osmotic pressure in watermelon fruit, so sugar accumulation was not related to seeds.