The experiments performed showed that the test allowed rapid and relevant prediction of red hair color based on the analysis of biological traces. The test also simultaneously enabled sex determination. This optimized test may also be useful in studies performed in other fields, such as dermatology. The study used 390 samples of buccal swabs taken from unrelated southern Polish Caucasians. The validation study was performed on different types of biological material analyzed in forensic science. DNA was extracted by either the silica-based extraction protocol or with Biorobot M48. DNA concentration was measured with Quanti-IT PicoGreen dsDNA Quantitation Kit. In addition to describing the samples tested, this paper also describes the assay design, with attention to multiplex PCR and minisequencing reactions. The description of the validation study addresses reliability, sensitivity, reproducibility, and biological traces. The description of the statistical evaluation of the influence of the MC1R genotype on phenotypic features focuses on the use of a causal Bayesian network that consisted of four nodes: “MC1R,” “individual hair color,” “population stratification,” and “other reason.” 4 tables, 7 figures, and 24 references