The chamois, Rupicapra spp, is a species in the family Bovidae and one of the most common mountain ungulates, distributed in the mountain ranges of Europe and Asia Minor, where it occurs in two species according to its morphological and behavioral characteristics: the Northern chamois R. rupicapra (with subspecies cartusiana, rupicapra, tatrica, balcanica, carpatica, asiatica and caucasica) and the Southern chamois R. pyrenaica (with subspecies parva, pyrenaica and ornata). The major histocompatibility complex (MHC) is a family of genes that encode receptors that recognize and bind antigens to present them to T cells. They are therefore central to vertebrate adaptive immunity. The MHC region comprises some of the most variable loci in the vertebrate genome, which have been associated with various fitness traits and thus with the long-term persistence of populations. Because of their wellcharacterized function and exceptional diversity, they represent excellent markers for evolutionary ecology and conservation. Traditional methods have been commonly used for their genotyping, but the introduction of next-generation sequencing has enabled more accurate and reproducible genotyping of such polymorphic gene families. In this work, the genetic diversity of the second exon of the MHC class II DRB locus was analyzed in 110 individuals from populations covering most of the range of the genus Rupicapra using a next-generation approach (Ion Torrent S5, Thermo Fisher). MHC DRB exon 2 encodes functionally important residues of the antigen binding groove and can therefore be used as a measure of the functional diversity of DRB alleles. Twenty-five MHC DRB alleles were found, each translated into a unique amino acid sequence, indicating the functional importance of polymorphism between alleles. Fourteen novel DRB alleles were identified in this study, two of which were found only in R. r. carpatica, two in R. r. asiatica, three in R. r. balcanica, and four only in R. r. rupicapra. A gene duplication was not identified. The high ratio of the relative rates of non-synonymous to synonymous mutations and the presence of trans-species polymorphisms suggest that this locus was under strong balancing selection throughout the evolutionary history of this species.