High-level resistance to aminoglycosides mediated by the production of 16S rRNA methylase among various gram-negative pathogens has been increasingly reported. Six 16S rRNA methylase enzymes have been identified: RmtA to RmtD, ArmA, and NpmA. We recently described the emergence of ArmA among Acinetobacter baumannii strains in North America. Here, we report the isolation of an Escherichia coli strain coproducing 16S rRNA methylase RmtB and extended-spectrum β-lactamase (ESBL) CTX-M-65 from an ambulatory female with a history of sickle-cell anemia in the United States. E. coli grew in a urine culture ordered as part of her evaluation for chronic kidney disease. This is the first instance in which RmtB has been shown to be encoded on the same plasmid as an ESBL.