A study was carried out using a set of four candidate gene markers for grain iron accumulation and genetic polymorphism was examined in twelve genotype of rice. The objective was to examine the nature of genetic variation at molecular level for characterization of these genotype. Putative candidate gene based primer directed amplification was successfully achieved by all the primer pairs. Polymorphism was recognized among the genotype in the form of absence of bands. Analysis of polymorphism pattern based on amplification profiles clearly indicated that nucleotide sequence variations at the primer binding sites within the putative candidate genes was the most probable casual factor for differential grain iron accumulation rice genotypes. Candidate gene based amplification assay dependent detection of non-alleles in some genotypes basically provided the basis to relate nucleotide variation at primer binding sites with differential grain iron accumulation in rice genotype.