© 2018 Elsevier B.V. Drylot beef cow-calf systems are viable alternative management systems to traditional forage-based systems. In such confinement, adding ionophores such as monensin to the diets of beef cattle is common, and has been shown to improve feed efficiency and increase average daily gain. The addition of monensin is also commonly utilized as a strategy for methane mitigation, as this ionophore class antimicrobial acts to interfere with ion flux primarily within Gram-positive cells through its action as an ion carrier. It is widely accepted that suppression of these ruminal organisms results in the reduction of substrates for rumen methanogenic archaea, reducing methane production. However, several studies have indicated that cattle may adapt to monensin, and within weeks of feeding, may return to prior levels of methane production. Our hypothesis is that feeding monensin to bred beef heifers in confinement will temporarily decrease methane production from shifts in the methanogenic archaeal and bacterial communities. Sixteen fall-born bred heifers were randomly assigned to 2 treatment groups (n = 8 per treatment) and were fed a control diet or a diet containing monensin for 70 days using headgates. In vivo gas exchange of oxygen consumption, carbon dioxide and methane production were measured for 24-h periods throughout the trial using individual calorimeter head boxes. Rumen content sampling was conducted on day 0, 18 and 53 of the trial through oral lavage. Upon completion of sampling, DNA was isolated for ruminal bacteriome composition utilizing deep, next-generation sequencing of the V1-V3 hypervariable regions of the 16S bacterial rRNA gene. Level of methanogen 16S rRNA was quantified using qPCR. There was a significant reduction in phylum SR1 (P < 0.05). The abundance of several OTUs was reduced between treatment with monensin, including Anaerofustis (P < 0.0001), Shuttleworthia (P < 0.0001) and Order Bacteroidales (P = 0.003). No significant shifts in key ruminal methanogenic archaeal groups as a percentage of total methanogen 16S rRNA occurred (P > 0.05). Heifers fed monensin did not produce significantly less methane than the control (P > 0.05) on a liters per day basis, which was consistent throughout the study. These data suggest methane production is not reduced long-term when animals are fed monensin in confinement. Additionally, the data suggest that monensin supplementation does not suppress all classical Gram-positive populations, rather it influences finer shifts in bacterial species that may be key in ruminant function. Determining the stability of the ruminal microbiome over time in heifers fed monensin may provide further insights to long-term methane mitigation in cow-calf systems.