Fermentation of dietary nutrients in the gastrointestinal (GI) tract of ruminants is a key mechanism utilized to meet daily energy requirements. Especially in lactating dairy cows, the GI microbiome plays a major role in the breakdown of indigestible plant polysaccharides and supply most AAs, fatty acids, and gluconeogenic precursors for milk synthesis. Although the contribution of the rumen microbiome to production efficiency in dairy cows has been widely researched over the years, variations throughout the lactation and lower gut microbiome importance to these traits remains poorly characterized. Therefore, we investigated throughout lactation the relationship between the rumen and lower gut microbiomes with production efficiency traits in Holstein cows. We found that the microbiome from both locations have temporal stability throughout lactation, yet factors such as feed intake levels played a major role in shaping microbiome diversity. The rumen microbiome had a composition dependent on feed intake level, while the lower gut microbiome was more associated with a potential enhanced ability to digest dietary nutrients. Therefore, association and prediction of milk production traits may be more correlated with microorganisms present in the lower gut. The findings of the current study are key elements to support more efficient and sustainable livestock production.