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Introduction: Beef is the most consumed red meat in the United States, and the US is the largest producer and consumer of beef cattle globally. Feed is one of the largest input costs for the beef cattle industry, accounting for 40–60% of the total input costs. Identifying methods for improving feed efficiency in beef cattle herds could result in decreased cost to both producers and consumers, as well as increased animal protein available for global consumption. Methods: In this study, rumen fluid was collected from low- (n = 14) and high-RFI (n = 15) steers. Rumen fluid was filtered through a 0.22 µM syringe filter, extracted using 0.1% formic acid in acetonitrile:water:methanol (2:2:1) and injected into the Dionex UltiMate 3000 UHPLC system with an Exactive Plus Orbitrap MS. Peaks were identified using MAVEN and analyzed using MetaboAnalyst 4.0 and SAS. Significance was determined using an α ≤ 0.05. Results: Eight metabolites were greater in low-RFI steers compared to high-RFI steers, including 3,4-dihydroxyphenylacetate, 4-pyridoxate, citraconate, hypoxanthine, succinate/methylmalonate, thymine, uracil, and xylose (P ≤ 0.05). These metabolites were predominantly involved in amino acid and lipid metabolism. Conclusions: Rumen fluid metabolomes differ in steers of varying feed efficiencies. These metabolites may be used as biomarkers of feed efficiency, and may provide insight as to factors contributing to differences in feed efficiency that may be exploited to improve feed efficiency in beef cattle herds. [ABSTRACT FROM AUTHOR]