The major histocompatibility complex (MHC) contains the most variable functional genes described in vertebrates. Individuals from natural populations deal constantly with a diverse range of pathogens and the polymorphism at MHC loci is what determines the diversity of foreign antigens that the host immune system can recognize. Polymorphism at individual loci may result in variable MHC class II (MHC-II) haplotypes. We characterized for the first time, the allelic diversity at the second DRB locus of the MHC-II in the Iberian red deer Cervus elaphus hispanicus. The studied population was sampled from a fenced estate that has been managed for hunting purposes and may provide information of the effect of game management on the genetic diversity of this species. Deer presented high levels of variation at MHC-II DRB-2 with 18 different alleles detected in 94 individuals. However, a significant heterozygous deficiency was found for MHC-II DRB-2 locus (92.5% of individuals only amplified one allele), whereas genotype frequencies at three neutral microsatellite loci were according to Hardy–Weinberg equilibrium, with heterozygosity over 50%. The analysis of control Iberian red deer from different geographic locations identified two expressed DRB-2 loci with a high degree of heterozygosis. The annual diversity index of MHC-II DRB-2 alleles significantly decreased along the 16-year study period, which was confirmed with losses in microsatellite markers. Although we cannot exclude positive non-assortative mating and/or substructured breeding (Wahlund effect) within our reference population, such unexpected apparent homozygosity at MHC DRB-2 loci is suggestive of null alleles occurring at our population. The observed pattern could be the result of a founder effect in this recently established population. Nonetheless, the loss of MHC-II DRB-2 allele diversity could reflect the effects of inbreeding in this fenced population managed for hunting. These findings support the importance of immunogenetic studies to assess management decisions, especially in isolated ungulate populations.