D16S543 is a complex STR locus consisting of five types of repeat units. The frequency distribution and genetic characteristics of this locus in Japanese were investigated using blood samples from 124 unrelated Japanese and 15 families. Alleles were detected using denatured polyacrylamide gels followed by automated analysis on an ABI 373 sequencer using Genescan software 672. Twenty-one alleles were identified, ranging in size from 281 to 489 bp. An allelic ladder containing the 21 alleles was constructed and used as a typing standard. The repeat unit arrays allowed the 21 alleles to be classified into three distinct groups, including alleles 1 to 7 in group I, alleles 8 to 14 in group II, and alleles 15 to 22 in group III. The alleles in group II were characterized by the insertion of one repeat unit of CAGG, one of AAAG, and three of AAGG, while the group III alleles differed from those of groups I and II by the insertion of a total of 32 repeat units ranging in 5 types. Within each group, the alleles differed from each other only in one 5′ side tetranucleotide AAGG. The power of discrimination (Pd) and the estimated heterozygosity were calculated to be 0.989 and 0.934, respectively. Typing of this locus was successfully applied in four old forensic materials. The study presented herein demonstrates that D16S543 is a highly polymorphic and applicable locus in Japanese.