ABSTRACT: During an extensive multipopulation study with Y-short tandem repeat (STR) loci, amplified using the AmpFℓSTR® Yfiler™ PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N=3141). By direct sequencing of this fragment, it was 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 fragment. 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 populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y-STR loci typeable using the AmpFℓSTR® Yfiler™ PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFℓSTR Yfiler kit even in the presence of female DNA containing the G variant.