We analyzed variation at the Y-linked DYS391 locus and its X homolog (DXYS391) in a sample from 347 unrelated individuals in the Korean population. The Y microsatellite locus and the homologous locus in the X chromosome revealed to have high levels of identity in their flanking regions that differ primarily by the presence of a (GACA)3 motif in the Y locus. The allele frequencies of DXYS391 and DYS391 were determined by a novel multiplex PCR amplification method using flanking primers, followed by denaturing polyacrylamide gel electrophoresis with allelic ladders. The Korean population surveyed here were found to be polymorphic and the allele 10 was present at the highest frequencies for both DXYS391 and DYS391 loci. The gene diversity values were 0.379 and 0.351 for the DXYS391 and DYS391 loci, respectively. Therefore, our results may provide information for one more example of similarity outside the pseudoautosomal regions between the dimorphic human sex chromosomes and can also be of useful application for investigating the statistical parameters of forensic interest.