In a multipopulation study with Y-short tandem repeat (STR) loci, amplified with the AmpFlSTR Y filer PCR amplification kit, the amplification of a 71 bp fragment was observed in 2.32 percent of the male samples (n=3,141). Using direct sequencing to this fragment, researchers determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single-nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragments. The SNP is located within an X-Y homologous region at Xq21.31 and was observed with the highest frequency within the African-American and Sub-Saharan African population. The presence of SNP on the X chromosome did not undermine the reliability of typing the DYS456 locus and the other Y-STR loci that were typed with the AmpFlSTR Yfiler PCR amplification kit. The study findings show that the presence of the 71 nt fragment has no impact on the interpretation of a Y-STR haplotype in males. In the presence of 4,000-fold excess female DNA that carries the SNP, 125 pg of DNA were typed correctly at the DYS456 locus as well as at all other Y-STR loci. In samples that contained male DNA only, this dinucleotide repeat on the X chromosome can be used as an additional marker. A description of materials and methods addresses the characteristics of the samples, PCR amplification, sequencing, allele-specific primer extension assay, and the male-female mixture experiments. 4 figures, 2 tables, and 25 references