Hair shaft is one kind of important biological evidence and is widely collected in the crime scene. However,it is very difficult to obtain full STR profiles as the nuclear DNA of hair shaft has degraded seriously. Mitochondrial DNA examination is the routine method for hair shaft,but it can not be used to identify individuals. Therefore,it is important to explore more genetic information of hair shaft in order to offer more clues for crime investigation.Protein is chemically more robust than DNA and can persist for a longer period.Proteins also contains genetic variation in the form of single amino acid polymorphisms(SAPs),which can be used to infer the status of non-synonymous single nucleotide polymorphisms(nsSNPs)for forensic ancestry analysis.Here,we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in104Chinese Han subjects. A total of552nsSNPs were imputed from703SAPs in460proteins.88nsSNPs were selected according to a call rate of15%,then validated using Sanger sequencing.Finally,clustering analysis and population random match probability calculation were performed to infer the ancestry of19Han Chinese individuals,and individual genotypes of1000genome populations were employed as reference data.The results demonstrated that nsSNPs imputed from hair shaft can be used to distinguish East Asian,European and African population.