The major drawback of a dioecious spine gourd (Momordica dioica Roxb.) is distinguishing whether the plant is male or female at an earlier development stage. An investigation was aimed to eliminate aforesaid limitation by identifying and validating sex linked molecular marker. Twenty six male and female genotypes of each sex were screened with 12 specific SSR primers. Only two SSR primers, MdSSR 5 (MADS-box gene of spine gourd) and CMbSSR 6 (MADS-box gene of cucumber) showed amplification and polymorphism in female genotypes only. These specific female linked markers manifest existent of non-epigenetic factors entailed in gender development in the spine gourd.