show Abstracthide AbstractThis study presents a complete DNA extraction process for keratinized materials, including sample washing, DNA release, and DNA purification and enrichment. Additionally, specific treatment was carried out in the library construction to increase the percentage of target sequences. We systematically evaluated keratinized materials of various types, ages, species, and preservation environments. The results demonstrate that this method is highly effective in enriching small fragments of DNA while also being simple and cost-effective. The quality of keratin DNA differs between types of keratin material and is greatly influenced by the number of years of storage and the environment, especially in hair samples where the longer the storage and the more tanning the DNA is damaged. We developed a standard process for applying keratinized DNA analysis and analyzed the reliability of its genetic information. Quality control aids were developed based on the re-sequencing results to facilitate better screening of available keratinized material. This study enhances the overall understanding of the genomes of keratinized materials and expands the source of samples for wildlife research.