Bovine leukocyte antigen (BoLA) DRB3 is a highly polymorphic gene in major histocompatibility complex(MHC) class II that plays a central role in immune responses and production factors. As of yet, molecular andevolutionary characteristics of BoLA-DRB3.2* have not been as fully understood as human and mouse.Therefore, we attempted to analyze variability and phylogeny of BoLA-DRB3.2* and illustrate some novelpractical evidence on interspecies diversity, the resistance /susceptibility points in cattle breeding, and vaccinedesign. Initially, BoLA-DRB3.2* alleles and orthologous exons in the selected livestock were retrieved andchecked. In the next step, the secondary/tertiary structure of BoLA-DRB3.2*24 gene product was modeled andvalidated. Then, hypervariable regions (HVRs) of alleles were identified by hybrid approaches. In the last step,interspecies relationship, allele’s phylogeny/grouping, and estimate of average evolutionary divergence wereexplored. Shannon entropy variation analysis showed eight HVRs and three semi-variable regions in BoLADRB3.2*alleles. These HVRs were present in all the three sub-structures and dominantly existed in alpha helix.In addition, strong relationships and little diversity were noted in phylogenetic trees of cattle, buffaloes, sheep,and goats. Furthermore, there was some evidence on divergence of DRB3 before speciation among thementioned species and possibility of cross prediction resistance/susceptibility alleles. Finally, DRB3 alleles weregrouped into seven clusters, and older and newer alleles were identified. The results show that similar studiesshould be done in other animals to better understand the nature of the DRB3 attributes.