Recently, the field of predicting phenotypes of externally visible characteristics (EVCs) from DNA genotypes with the final aim of concentrating police investigations to find persons completely unknown to investigating authorities, also referred to as Forensic DNA Phenotyping (FDP), has started to become established in forensic biology. The authors previously developed and forensically validated the IrisPlex system for accurate prediction of blue and brown eye color from DNA, and recently showed that all major hair color categories are predictable from carefully selected DNA markers. HIrisPlex consists of a single multiplex assay targeting 24 eye and hair color predictive DNA variants including all 6 IrisPlex SNPs, as well as two prediction models, a newly developed model for hair color categories and shade, and the previously developed IrisPlex model for eye color. The HIrisPlex assay was designed to cope with low amounts of template DNA, as well as degraded DNA, and preliminary sensitivity testing revealed full DNA profiles down to 63pg input DNA. The power of the HIrisPlex system to predict hair color was assessed in 1,551 individuals from three different parts of Europe showing different hair color frequencies. Using a 20 percent subset of individuals, while 80 percent were used for model building, the individual-based prediction accuracies employing a prediction-guided approach were 69.5 percent for blond, 78.5 percent for brown, 80 percent for red and 87.5 percent for black hair color on average. Results from HIrisPlex analysis on worldwide DNA samples imply that HIrisPlex hair colour prediction is reliable independent of bio-geographic ancestry (similar to previous IrisPlex findings for eye colour). The authors furthermore demonstrate that it is possible to infer with a prediction accuracy of >86 percent if a brown-eyed, black-haired individual is of non-European (excluding regions nearby Europe) versus European (including nearby regions) bio-geographic origin solely from the strength of HIrisPlex eye and hair color probabilities, which can provide extra intelligence for future forensic applications. The HIrisPlex system introduced here, including a single multiplex test assay, an interactive tool and prediction guide, and recommendations for reporting final outcomes, represents the first tool for simultaneously establishing categorical eye and hair color of a person from DNA. The practical forensic application of the HIrisPlex system is expected to benefit cases where other avenues of investigation, including STR profiling, provide no leads on who the unknown crime scene sample donor or the unknown missing person might be. (Published Abstract)