The interplay between host genotype and commensal microbiota at different body sites can have implications for health and disease. In dairy cows, polymorphism of bovine major histocompatibility complex (BoLA) gene has been associated with mastitis susceptibility. However, mechanisms underlying this association are as yet poorly understood. In the present proof-of-concept study, we sought to explore the link between polymorphism of BoLA genes and the dynamics of commensal microbiota of the mammary gland (MG) at early stages of lactation. To this end, 63 Holstein dairy cows were subjected to longitudinal aseptic milk sampling during the first week of lactation (composite colostrum/milk samples were collected from each cow at d 0, 2, and 6 relative to calving). Microbiota profiling was performed using high-throughput sequencing of the V1-V2 regions of the bacterial 16S rRNA genes and ITS2 region of the fungal ribosomal DNA. Polymorphism of BoLA genes were determined using PCR-RFLP (Bst YI) of the BoLA-DRB3 exon 2. In general, transition from colostrum to fresh milk resulted in gradual but distinct shifts in the compositions of both bacterial and fungal communities of the MG. In addition, comparison of the composition of microbiota among the identified BoLA-DRB3 haplotypes (n = 3) revealed distinct clustering patterns (P_PERMANOVA < 0.05) for both bacterial and fungal communities at d 0, whereas this effect was less pronounced on the composition of microbiota of subsequent milk samples (d 2 and 6; P_PERMANOVA > 0.05). At d 0, each BoLA-DRB3 haplotype resulted in enrichment of several operational taxonomic units (OTUs) within colostrum microbiota, with the most notable differences being observed for certain coagulase-negative staphylococci (CNS), namely S. chromogenes, S. sciuri, and S. succinus. The role of CNS species in modulating MG homeostasis is controversial; some species are considered as the most important causes of subclinical mastitis during the periparturient period while other species have been reported to protect MG against invasion by major mastitis pathogens. Overall, the present study provides novel insights into the role of BoLA gene in determining the dynamics of MG microbiota during early stages of lactation, and potential implications that it may have for MG health and mastitis susceptibility.