Asclepiadaceae is an economically important family species which are the source of fiber, rubber and dyes. In this study, genetic variability has been determined in three species, that is, Tylophora hirsuta, Wattakaka volubilis and Cryptolepis buchananii. The aim of present study was to understand the extent and pattern of genetic diversity among the individuals of same and different species of Asclepiadaceae. To asses the level of polymorphism within the species and members of different species, randomly amplified polymorphic DNA (RAPD) markers were used. Sixty RAPD primers of OPA, OPC, OPF and OPG series were used; only eight primers of OPC series gave amplification. Maximum polymorphism at interspecific and intraspecific levels was shown by OPC 09 and minimum polymorphism was observed in OPC 05. The data was analyzed using software numerical taxonomy system (NTSYS) cluster analysis PC version 2.20. In total 190 monomorphic and 78 polymorphic bands were produced from all primers. Therefore, out of 322 amplified products, 59% were monomorphic and 24.22% were polymorphic. Low genetic diversification was found both at intraspecific and interspecific level. Mixed pattern of grouping in the analyses indicated the close affinities of species with each other.