STR multiplexes have been indispensable for the efficient genotyping of forensic samples. The PowerPlex® 16 System contains the core CODIS loci, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, the sex determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. This multiplex satisfies the locus requirements for most national databases and is the most efficient currently available system due to its single PCR amplification. To provide the groundwork for judicial acceptance, including the publication of primer sequences, and to evaluate laboratory-to-laboratory variation, a developmental validation for casework on this commercially available system was performed in 24 laboratories and produced the following conclusions. Amplification was reliable on a variety of thermal cyclers and product could be analyzed on either an ABI PRISM® 310 Genetic Analyzer or an ABI PRISM® 377 DNA Sequencer. Genotyping using single source samples was consistent between 0.25 and 2 ng of input DNA template with a few laboratories obtaining complete genotypes at 0.0625 ng. However, heterozygote allele imbalance (_60% peak height balance) caused by stochastic effects was observed at a rate of 13% with 0.125 ng DNA and 22% at 0.0625 ng DNA. Mixture analyses were done using a total of 1 ng of DNA template. Most alleles were detected in mixtures of 4 to 1 and some minor alleles were detected in mixtures of 19 to 1. Optimum amplification cycle number was dependent on the sensitivity of the detection instrument used and could also be adjusted to accommodate larger amounts of DNA on solid supports such as FTA® paper. Reaction conditions including volume, annealing temperature, and concentrations of primer, AmpliTaq Gold®, and magnesium were shown to be optimal yet robust enough to withstand moderate variations without affecting genotype analysis. Environmental, matrix and standard source analyses revealed an ability to obtain complete genotypes in all sample types except those exposed to 80°C for 12–48 days. Finally, comparison of genotype results from the PowerPlex® 16 System with other commercially available systems on non-probative reference and forensic samples showed consistent results.