Most rumen bacteria are uncultured, making it hard to identify their niche and what feed carbohydrates they consume. Our objective was to identify uncultured rumen bacteria that use one feed carbohydrate (glucose) by (1) incubating bacteria in a fluorescent glucose compound (2-NBDG), (2) using cell sorting to separate out cells that fluoresce, and (3) using high-throughput DNA sequencing to identify sorted cells. Mixed bacteria readily took up 2-NBDG, and flow cytometry showed that 10.0 (0.5 SEM) % cells became fluorescent (positive) (n = 18). Positive cells were separated with fluorescence-activated cell sorting, and post-sort analysis revealed sorted cells were highly [94.9 (0.3 SEM) %] pure (n = 18). The 16S ribosomal RNA gene of sorted cells was sequenced using the Illumina MiSeq platform. This sequencing revealed several bacteria related to cultured groups known to use glucose, including Prevotella, Butyrivibrio, and Succinivibrio. However, many bacteria belonged to uncultured groups, including those of family BS11, class koll 11, and order RF 39. Control experiments confirmed that RF 39 and other uncultured groups indeed cannot be easily cultured, as they were not detected among cells grown on media including glucose as the sole carbon source. Importantly, RF 39 has been previously associated with efficiency of milk production. Our work begins to reveal the niche of this group by showing some members consume glucose. Work is ongoing to synthesize and test other fluorescent glucose compounds. Our work supports that 2-NBDG, in combination with other compounds, could be used to identify which uncultured bacteria consume which feed carbohydrates, helping define what role uncultured bacteria play in the host.