DNA was extracted from human samples and bisulfite modified using commercial bisulfite modification kits. Specific primers were used to amplify the region of interest, and the methylation profile of the CpG sites were determined by pyrosequencing. The percent methylation values at each CpG site were determined in multiple samples and averaged for each tissue type. The versatility of these new markers is presented by showing the results of validation studies on sensitivity, human specificity, stability and mixture resolution. When testing the markers using different organisms, researchers obtained positive results for certain non-human primate samples; however, all other tested species were negative. The lowest concentration consistently detected varied from 0.1 to 10 ng, depending on the locus, indicating the importance of primer design and sequence in the assay. The method also proved to be effective when inhibitors were present in the samples or when samples were degraded by heat. Simulated case samples were also tested. In the case of mixtures of different cell types, the overall methylation values varied in a consistent and predictable manner when multiple cell types were present in the same sample. Overall, the validation studies demonstrate the robustness and effectiveness of this new tool for body fluid identification. (Publisher abstract modified)