In order to evaluate the forensic applicability of the STR locus D20S161 and construct a preliminary database, the genotype distributions and allele frequencies in five populations from three main ethnic groups were investigated, including Germans, Slovakians, African Americans, Japanese and Chinese. A total of 512 samples from unrelated individuals and 85 confirmed father/mother/ child triplets were analyzed by PCR and allele determination was carried out by comparison with a sequenced human allelic ladder. The results showed that D20S161 typing was both precise and reliable. A total of 7 alleles was found in these populations and no evidence of deviation from Hardy-Weinberg equilibrium was observed. Pairwise comparisons between populations showed that there were significant differences in the distributions of the allele frequencies among the three main ethnic groups. No mutation events were observed from the confirmed father/mother/child triplets. With a maximum likelihood method, the mutation rate was indirectly estimated as 2.5 × 10–5. These results suggest that D20S161 is a useful marker for forensic casework and paternity analysis.