The clinical usefulness of aminoglycosides has been revisited as an effective choice against β-lactam-resistant and fluoroquinolone-resistant gram-negative bacterial infections. Plazomicin, a next-generation aminoglycoside, was introduced for the treatment of complicated urinary tract infections and acute pyelonephritis. In contrast, bacteria have resisted aminoglycosides, including plazomicin, by producing 16S ribosomal RNA (rRNA) methyltransferases (MTases) that confer high-level and broad-range aminoglycoside resistance. Aminoglycoside-resistant 16S rRNA MTase-producing gram-negative pathogens are widespread in various settings and are becoming a grave concern. This article provides up-to-date information with a focus on aminoglycoside-resistant 16S rRNA MTases.