Biochemical research was carried out on 4 human skeletal remains from historical lime-layered tombs assigned to the Joseon Dynasty in Oknam-ri, Seocheon. The preservation of femur was evaluated by stereoscopic microscopy and scanning electron microscopy. Most of specimens showed good histological preservation. The histological results proved to be a good potentiality for biochemical analysis using bio-molecules. The amelogenin gene and mitochondrial DNA (mtDNA) analyses revealed that three specimens perhaps have maternal consanguinity due to sharing with mtDNA haplogroup D4b1, and two specimens buried in the same tomb were a couple in Gatjaegol site. Carbon and nitrogen stable isotope analysis indicated that four deads diet were built around C3 plant as rice, barley, wheat and bean. In this study we characterized genetic and diet features from the social stratum who could make lime-layered tombs during period of the Joseon Dynasty. The results suggest that biochemical research using the human skeletal remains from the Joseon Dynasty has the great potential and reasonable value for archaeology, anthropology, and population genetics.