Abstract: A short tandem repeat multiplex assay has been successfully developed with 25 autosomal loci plus the sex-typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present were distributed from 65 base pairs (bp) to less than 400 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the successful amplification of a wide range of genomic DNA sample concentrations from 2 ng to as low as 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.