The RAPD analysis was carried out with 28 crosses, 8 parents and 2 checks of opium poppy. Purified and isolated DNA was subjected to PCR based marker analysis (RAPD) for assessment of genetic diversity. The quality of DNA was determined by calculating ratio between A260 and A280 observed between 1.857 to 2.167 which indicated a good quality of plant DNA. The concentration of DNA ranged between 123 μg/μl (UOP-60 x UOP- 99) to 750 μg/μl (UOP-79 x UOP- 80). In the RAPD analysis 12 primers gave good amplified products with template DNA. Polymorphism shown by 12 primers ranged between 50 per cent (OPP-02) to 100 per cent (OPA-01, OPA-08, OPB-06 and OPD-05). Average polymorphism was found to be 84.80 per cent. From RAPD profiling similarly matrix was obtained and Jaccard’s similarity coefficient lies between 0.57 (UOP-69 x UOP-80) to 0.95 (UOP-53 x UOP-79) with an average of 0.79. On this basis a dendrogram was constructed using UPGMA method. Dendrogram differentiated 28 crosses, 8 parents and 2 checks of opium poppy into one major and four minor groups. Further genetically diverse parents and crosses can be alternatively used for accumulating favourable genes so as to finally improve the productivity.