The aim of this study is to assess the utility of the STR D5S373 in human identification. PCR amplification and electrophoretic separation were optimized in order to achieve unambiguous phenotyping. We concluded that primer concentration and annealing temperature are the main factors affecting the specificity of PCR. In our population survey including three human major groups (Europe, Sub-Saharan Africa, and Asia), up to six alleles and six interalleles have been found ranging in size from 86 to 101 bp. The phenotypes were determined using horizontal polyacrylamide gel electrophoresis, a technique which has turned out to be suitable for separating fragments as close as 1 bp. In each population, the genotype frequencies conformed to the expectations of genetic equilibrium. Sequence studies were carried out to make the allele nomenclature fit to ISFH recommendations. Results from our population analysis of D5S373 show clear differences in allelic frequency patterns among the three major human groups examined. Human identification parameters estimated from our study are similar to those obtained for other STRs currently used in DNA testing.