DNA analysis of ancestry informative markers (AIMs) and physical trait markers from biological stains can help provide investigative leads in cases without suspects. To enhance the resolution and informative value of two previously developed single nucleotide polymorphism (SNP) multiplexes, the 34-plex and Eurasiaplex assays (Phillips et al. [1], Phillips et al. [2]) differentiating European, South Asian and Middle East populations, we have selected an additional 22 AIM-SNPs. The selected markers focus on the differentiation of Europeans and Asians and we supplemented this AIM set with 10 recently published pigmentation markers informative for eye, hair and skin colour (Walsh et al. [4], Sturm [3]). Comparisons of reference SNP data from HGDP-CEPH and 1000 Genomes population panels with 7 Eurasian study populations were made using STRUCTURE and principal component analysis (PCA) indicating that this multiplex can improve the differentiation of populations within East Asia. In a small pilot study using voluntary donors from Turkey, the IrisPlex SNP markers (Walsh et al. [4]) built into our multiplex were successfully used to make predictions about eye colour.