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Background: The major histocompatibility complex (MHC) is responsible for detecting and addressing foreign pathogens inside the body. While the general structure of MHC genes is relatively well conserved among mammalian species, it is notably different among ruminants due to a chromosomal inversion that splits MHC type II genes into two subregions (IIa, IIb). Recombination rates are reportedly high between these subregions, and a lack of linkage has been documented in domestic ruminants. However, no study has yet examined the degree of linkage between these subregions in a wild ruminant. The white-tailed deer (Odocoileus virginianus), a popular ruminant of the Cervidae family, is habitually plagued by pathogens in its natural environment (e.g. Haemonchus contortus, Elaeophora). Due to the association between MHC haplotypes and disease susceptibility, a deeper understanding of MHC polymorphism and linkage between MHC genes can further aid in this species' successful management. We sequenced MHC-DRB exon 2 (IIa) and MHC-DOB exon 2 (IIb) on the MiSeq platform from an enclosed white-tailed deer population located in Alabama. Results: We identified 12 new MHC-DRB alleles, and resampled 7 alleles, which along with other published alleles brings the total number of documented alleles in white-tailed deer to 30 for MHC-DRB exon 2. The first examination of MHC-DOB in white-tailed deer found significantly less polymorphism (11 alleles), as was expected of a non-classical MHC gene. While MHC-DRB was found to be under positive, diversifying selection, MHC-DOB was found to be under purifying selection for white-tailed deer. We found no significant linkage disequilibrium between MHC-DRB and MHC-DOB, suggesting that these loci are unlikely to be closely linked. Conclusions: Overall, this study identified 12 new MHC-DRB exon 2 alleles and characterized a new, non-classical, MHC II gene (MHC-DOB) for white-tailed deer. We also found a lack of significant linkage between these two loci, which supports previous findings of a chromosomal inversion within the MHC type II gene region in ruminants, and suggests that white-tailed deer may have a recombination hotspot between these MHC regions similar to that found for Bos taurus. [ABSTRACT FROM AUTHOR]