The extensive use of aminoglycosides to treat bacterial infections has led to significant resistance, posing a global health threat. Recent clinical reports highlight high levels of aminoglycoside resistance due to Arm/Kam methyltransferases, which methylate specific nucleotides in 16S rRNA, preventing antibiotic binding to the ribosome. This study compared the ribosomal A site binding patterns of Arm methyltransferases from clinical pathogens (ArmA, RmtB, RmtC, and RmtD) with those of the Sgm methyltransferase from a natural aminoglycoside producer. We introduced single mutations near the G1405 nucleotide in helix 44 of 16S rRNA to assess their impact on the methylation ability of Arm methyltransferases in E. coli cells with homogeneous mutant ribosomes. We evaluated how these mutations affected bacterial viability in cells with mixed and homogeneous ribosome populations and determined the minimal inhibitory concentration of kanamycin to assess their impact on Arm enzyme activity. Notably, Sgm methyltransferase exhibited a distinct methylation pattern compared to Arm methyltransferases from clinical strains. Structural comparisons of Sgm, RmtB, and RmtC revealed different spatial orientations of key amino acids involved in ribosomal binding, highlighting evolutionary differences. This research enhances understanding of Arm methyltransferases and lays the groundwork for designing inhibitors to combat this potent form of antibiotic resistance.