DNA from 1020 unrelated male individuals sampled from five locations of Chile (Iquique, Santiago, Concepción, Temuco, and Punta Arenas) were typed for autosomal STRs, Y-STRs, and the mtDNA Control Region. The populations were selected to develop reference databases to support forensic casework and relationship testing. Allele frequencies for 15 autosomal STR loci across the five sampled sites were compiled. As there was considerable overlapping of birthplaces of subjects sampled from these five sites, the pooled dataset was re-grouped based on birthplaces of the subjects into eight geo-political birthplace regions of the country. Each of these populations was evaluated for conformance to Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between loci and within the populations was assessed. Descriptive statistics, i.e., power of discrimination (PD), power of exclusion (PE), and mean power of exclusion were determined. No deviations from HWE expectations (p < 0.05) and LD were detected. Combined PD and PE for each population exceeded 0.99999. Y-STR and mtDNA haplotype frequencies were developed and haplotype sharing within and between populations was evaluated. The PD for the Y-STR database is 0.99841 and for the mtDNA database it is 0.99356. Population substructure on the haplotype data evaluated by AMOVA indicated approximately 0.03% of the variation detected originated from differences among the eight birthplace regions. Independence between Y-STR haplotypes, mtDNA haplotypes, and autosomal loci was assessed using a mismatch distribution approach. © 2009.