Abstract: Using RAPD (random amplified polymorphic DNA) and SRAP (sequence-related amplified polymorphism) molecular markers technology, the DNA fingerprints of ‘Ruyu No.5’ bitter gourd and its parents were analyzed to obtain the different genes differentially expressed in order to identify the purity of hybrid seeds eventually. After repeated experiments, only 1 RAPD primer(R10) screened out from 52 could amplify the characteristic polymorphic bands of F₁ generation with its male parent and 2 SRAP primers (Me2-Em9, Me3-Em8) screened out from 144 could amplify the characteristic polymorphic bands of F₁ generation with its female parent. With the two markers from RADP (R10) and SRAP (Me2-Em9) possessing high stability and repeatability, the seed of ‘Ruyu No.5’ from Hexi Corridor in Gansu Province were identified, and the result was roughly according to the identification from field. The result showed that this method for low cost and easy operation can be perfect to identify the purity of ‘Ruyu No.5’ bitter gourd seeds.