Abstract: In this study, shoot regeneration using cotyledons derived from seedlings of diploid and triploid yellow watermelon (cultivars Hwang Fong Yellow Queen, Round Dragon and Chin San Seedless) was investigated. Multiple shoots from auxiliary meristems were obtained without adventitious shoot. Shoot regeneration system for watermelon was successfully established from cotyledon sections of 4 to 5 day-old in vitro seedling. The explants were collected from the proximal cotyledon with hypocotyls segment. Highest mean number of multiple shoots were obtained (9.83±0.54) for cultivar Hwang Fong Yellow Queen on MS medium supplemented with 20 μM BAP as compared to (8.87±0.81) for Chin San Seedless on 5 μM of BAP and (6.00±0.32) for Round Dragon on 10 μM of BAP. From these results, cultivar Hwang Fong Yellow Queen was used for further experiment due to its highest percentage of germination and mean number of shoots. Subsequently, the cultivar successfully rooted (80%) on half strength MS medium supplemented with 0.5 μM IAA. Finally, 50% of these rooting plantlets were acclimatized on soil.