Abstract: Estimation of genetic diversity of Brassica germplasm provides the basis for rapeseed/mustard genetic improvement. Studies were undertaken to estimate the genetic diversity of 30 lines of Brassica napus using Randomly Amplified Polymorphic DNA (RAPD) primers. A total of 30 B. napus genotypes of local and exotic origin were characterized using molecular markers. Four RAPD primers were used to estimate the genetic distances among the genotypes in all the possible combinations. The genetic diversity study revealed different levels of genetic polymorphism for RAPD primers GLA05, GLA07, GLA09 and GLA10, resulting in amplification of 5.7, 3.5, 3.1 and 5.4 scorable bands (loci) per genotype, respectively. Individual genetic distances observed among B. napus genotypes ranged from 6.5 to 51%. Bivariate data matrix was generated and genetic distances were calculated using Unweighted Pair Group of Arithmetic Mean (UPGMA) procedure. The UPGMA cluster analyses revealed maximum genetic dissimilarity for 8966-1 and 8969-1 genotypes, closely followed by Ganyou-5, 89127-1, 89111-2 and Mlep-048. It is recommended that among the thirty B. napus genotypes, genetically distinct lines pointed out in the present study, should be used in future breeding programs for improvement of Brassica napus.