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Ruminants are a critical human food source and are a potentially important source of global methane emissions. Because of their unique digestive physiology, ruminants rely upon a symbiotic relationship with complex and rich microbial communities in the foregut to allow digestion of complex carbohydrates. This study used 16S rRNA gene sequencing to investigate the composition of microbial communities of three rumen micro-environments (free fluid, the fibrous pack, and the rumen mucosa) from cattle fed identical diets. While a phylogenetic core including the most abundant and most common ruminal taxa (members of Bacteroidetes and Firmicutes) existed across micro-environments, their abundances differed significantly, and specific lineages were discriminant of individual micro-environments. Firmicutes, and specifically Ruminococcaceae, Lachnospiraceae, and Clostridium were discriminant of fluid communities, while Bacteroidales and Desulfovibrio were discriminant of mucosa-associated communities. A set of four more diverse lineages were discriminant of pack-associated communities that included Paraprevotellaceae, Succinivibrionaceae, RFP12 (Verrucomicrobia), and Treponema. Our findings indicate that different ecological niches within each micro-environment have resulted in significantly different microbial communities and provide baseline values for the diversity and community structure of microbial communities from rumen fluid, pack, and mucosa that can contextualize the influence of environmental factors such as diet.