The Altai Mountains have been a long-term boundary zone between the Eurasian Steppe populations and South and East Asian populations. To disentangle some of the historical population movements in this area, 14 ancient human specimens excavated in the westernmost part of the Mongolian Altai were studied. Thirteen of them were dated from the Middle to the End of the Bronze Age and one of them to the Eneolithic period. The environmental conditions encountered in this region led to the good preservation of DNA in the human remains. Therefore, a multi-markers approach was adopted for the genetic analysis of identity, ancestry and phenotype markers. Mitochondrial DNA analyses revealed that the ancient Altaians studied carried both Western (H, U, T) and Eastern (A, C, D) Eurasian lineages. In the same way, the patrilineal gene pool revealed the presence of different haplogroups (Q1a2a1-L54, R1a1a1b2-Z93 and C), probably marking different origins for the male paternal lineages. To go further in the search of the origin of these ancient specimens, phenotypical characters (i.e. hair and eye color) were determined. For this purpose, we adapted the HIrisPlex assay recently described to MALDI-TOF mass spectrometry. In addition, some ancestry informative markers were analyzed with this assay. The results revealed mixed phenotypes among this group confirming the probable admixed ancestry of the studied Altaian population at the Middle Bronze Age. The good results obtained from ancient DNA samples suggest that this approach might be relevant for forensic casework too.