Technical improvements in high-throughput sequencing technologies have opened new frontiers in microbiome research by allowing cost-effective characterization of complex microbial communities, including that of the bovine host. Targeted next generation sequencing has been shown to be an efficient approach for detection and identification of microorganisms and is more likely to be implemented in clinical and diagnostic settings due to its lower cost and shorter labor time. Such an approach relies on sequencing of a genetic marker, the 16S rRNA gene, for specific characterization of bacterial communities and bacterial pathogenic agents. Given the potential role of the microbiome in animal health and disease, the overall objectives of this dissertation were to: 1) identify the most appropriate DNA extraction protocol that efficiently isolates a majority of the heterogeneous bacterial species encountered in non- mastitic and mastitic milk samples for accurate taxonomic profiling and detection of clinical mastitis causative agents (Chapter two); 2) use high-throughput sequencing of the 16S rRNA gene to characterize the bovine microbiome of distinct anatomical sites (mammary gland and upper respiratory tract) and its associations with bovine health (Chapters three and four); and 3) investigate the origin of the bovine microbiome (Chapter five). The present dissertation describes the effective application of the16S rRNA gene amplicon sequencing in clinical science and discovery of new pathogens. The knowledge gained from this research is novel and serves as an anchor for exploring the unfolding frontier of knowledge about the bovine microbiome across various anatomical sites and its effect on animal health.