All but 1 locus of the additional 20 mini-short tandem repeat (miniSTR) markers was under 140 base pairs (bp). These new loci were run across 665 U.S. population samples, including African-Americans, Caucasians, and Hispanics. Population statistics were determined for each group. All 665 samples were resolved from one another with all 26 new loci. The heterozygosity values for all of these markers were comparable to those of the 13 core CODIS loci, with a few of the new marker values being lower. Allelic ladders were created for each of these new miniSTR loci using the population samples. Now that each of these miniSTR loci have been characterized and optimized, the goal is to create larger multiplexes, with several loci present in different dye sets, so as to maximize the effectiveness of these markers on reference samples. Even used as single markers, however, their contribution to the forensic community in assisting in the analysis of degraded DNA samples may prove useful, as well as providing additional discrimination in complex paternity cases or missing person cases. Many of these miniSTR markers are being currently used in laboratories across the world as supplements in order to obtain full profiles from samples that generate partial profiles using commercial STR kits. Descriptions of materials and methods address the selection of loci and primer design, the source of DNA samples for population testing, PCR amplification, analysis on the ABI 3100 and 3130xl Genetic Analyzer, the generation of allelic ladders and GeneMapper ID bins and panels, DNA sequencing, and data analysis. 5 tables, 2 figures, and 22 references