Based on the collection of Hainan Island landraces of Cucurbita moschata, the present study was aimed to analyze their genetic specificity and establish DNA fingerprinting for 28 landraces by Inter-simple sequence repeat(ISSR)and sequence-related amplified polymorphism (SRAP), which will provide applicable guidance for identification, evaluation, protection and utilization of Hainan Island landraces in C. moschata. The result showed that genetic specificity was significant among the 28 landraces with special genetic background, 10 specific markers and 11 unique-lack bands scored were produced by 6 ISSR primers and 11 SRAP primer pairs selected; Four DNA fingerprintings for the 28 landraces were developed by two ISSR primer combinations and two SRAP primer combinations, i.e. UBC807/UBC814/UBC844/UBC868, UBC808/UBC814/UBC844/UBC868, Me1/Em2+Me1/Em10+ Me2/Em3, Me1/Em1+Me1/Em10+Me8/Em3, respectively. All the DNA fingerprintings developed were intuitionistic and simple. ISSR and SRAP molecular markers could be effectively used in the analysis of genetic specificity and DNA fingerprinting establishment for the landraces of C. moschata.