In forensic toxicology, the abuse of various opiate preparations, such as raw opium and heroin, is of interest since the metabolic pathways of these opiates overlap. Although the pharmaco(toxico)kinetics in hair is not clearly understood, melanin is thought to play a key part in the incorporation and distribution of drugs and metabolites in hair. Therefore, in the present study, a simultaneous quantification method for the determination of codeine, morphine, norcodeine, normorphine and 6-acetylmorphine (6-AM) in hair was developed in order to analytically diagnose chronic users of opiates including morphine and codeine preparations, raw opium and heroin. Furthermore, the effect of hair pigmentation on the distribution of opiates in hair was investigated using lean Zucker rats with both dark grey and white hair on the same body. Opiates were extracted using 0.1 M hydrochloric acid followed by solid phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide and analyzed using gas chromatography/mass spectrometry (GC/MS). The method was fully validated and applied to the animal study. In conclusion, the current study demonstrates that codeine, morphine and their metabolites were successfully determined in both pigmented and non-pigmented hair. However, the melanin content plays an important role in the degree of incorporation of morphine, codeine and their metabolites into hair.