Abstract Groundnut yield can be further enhanced by improving pod and kernel size vis-a-vis mass. Marker assisted breeding will be an ideal option for directed improvement of hundred kernel mass. A study was undertaken to detect molecular diversity using 35 SSRs in 12 mutant genotypes, developed through chemical mutagenesis, from an interspecific large kernel size pre-breeding line and to identify markers associated with kernel mass. SSRs yielded an average of 3.57 polymorphic bands per primer. Average polymorphism and PIC were 64.95% and 0.62, respectively. Cluster analysis revealed two main clusters separated at 61% Jaccard's similarity coefficient. Vast of the genotypes were grouped into single cluster, confirming common pedigree of these genotypes. AMOVA among 12 mutant genotypes and their parent detected 15% of total variation associated with kernel mass. K-W ANOVA detected significant association of five SSRs with kernel mass. Among these associated primers, TC3A12 and TC9H09 accounted for 28% and 12% of phenotypic variation due to kernel mass and were associated with major QTLs. Out of these two associated primer, TC3A12 differentiated genotypes with higher kernel mass from genotypes with lower kernel mass by amplifying a band of approximately of 450bp. Thus association of TC3A12 primer with a major QTL of kernel mass was further validated in genotypes with diverse background. The TC3A12 primer discriminated genotypes with higher kernel mass from genotype with lower kernel mass by amplifying the band of 400bp among genotypes with higher kernel mass
