As a result of climatic, political, and economic changes, rice has become a major food crop in maintaining global food security. This study is designed to describe the phenotypic and grain quality, make molecular fingerprinting profiles, and assess the biodiversity using DNA barcoding for thirteen high-yielding genotypes. A variety of biochemical parameters and molecular marker techniques were used. The chemical composition was studied and labeled among the genotypes. The results using electronic imaging showed the phenotypic characteristics of rice grains, such as grain dimensions were all significantly different amongst studied genotypes. Furthermore, the SEM scan results revealed differences in the outer surface of brown rice (decorated rice) and grain coat (rough rice) for the studied genotypes. Eighteen start codon-targeted (SCoTs) primers were used to assess the biodiversity of the studied genotypes. Phylogenetic analysis classified the thirteen varieties into three clusters based on SCoTs data, revealing the wide biodiversity among them. DNA barcoding using matK and rpoC1 chloroplast genes was identified and sequences were registered with accession numbers in the NCBI.