This study was performed to analyze genetic relationship of the four major Cucurbitaceae crop. We used 120 Expressed Sequence Tag(EST)-Simple Sequence Repeat(SSR) primer sets of developed from watermelon and published in International Cucurbit Genomics Initiative (ICuGI) database. Among 120 EST-SSR primer, 51(49.17%) EST-SSR primer set successfully amplified and 49(40.8%) EST-SSR primer set showed polymorphisms among eight cultivars of Cucurbitaceae. In the first instance, amplified PCR products analysis was conducted by the agarose-gel electrophoresis then further analyzed by using Fragment Analyzer. A total 382 PCR band were producted by 49 EST-SSR primers in 24 plant panels, used the analysis of pairwise similarity and dendrogram construction. Assessment of the genetic relationships resulted in similarity index with range of 0.0103 to 0.8452. In dendrogram, 24 plant panels were formed three major groups (A, B, C) and 7 subgroups (A-1, A-2, B-1, B-2, B-3, C-1, C-2). Major group A was comprised of 2 subgroups, subgroup A-1 (6 watermelon cultivars, Citrullus lanatus var. vulgaris Schrad.) and subgroup A-2 (3 wild type watermelon, Citrullus lanatus var. citroides Mats. & Nakai). Major group B was comprised of 3 subgroups, subgroup B-1 (4 melon cultivars, Cucumis melo var. cantalupensis Naudin.), subgroup B-2 (2 oriental melon cultivars, Cucumis melo var. conomon Makino.) and subgroup B-3 (5 cucumber cultivars, Cucumis sativus L.). Major group C was comprised of 2 subgroups, subgroup C-1 [2 squash/ pumpkin cultivars, Cucurbita moschata (Duch. ex Lam.)/Duch. & Poir. and Cucurbita maxima Duch.] and subgroup C-2(2 squash/pumpkin cultivars, Cucurbita pepo L./Cucurbita ficifolia Bouche.)