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Aminoglycoside antimicrobials remain valuable therapeutic options, but their effectiveness has been threatened by the production of bacterial 16S ribosomal RNA methyltransferases (16S-RMTases). In this study, we evaluated the genomic epidemiology of 16S-RMTase genes among Gram-negative bacteria circulating in the American continent. A total of 4877 16S-RMTase sequences were identified mainly in <i>Enterobacterales</i> and nonfermenting Gram-negative bacilli isolated from humans, animals, foods, and the environment during 1931–2023. Most of the sequences identified were found in the United States, Brazil, Canada, and Mexico, and the prevalence of 16S-RMTase genes have increased in the last five years (2018–2022). The three species most frequently carrying 16S-RMTase genes were <i>Acinetobacter baummannii</i>, <i>Klebsiella pneumoniae</i>, and <i>Escherichia coli</i>. The <i>armA</i> gene was the most prevalent, but other 16S-RMTase genes (e.g., <i>rmtB</i>, <i>rmtE</i>, and <i>rmtF</i>) could be emerging backstage. More than 90% of 16S-RMTase sequences in the Americas were found in North American countries, and although the 16S-RMTase genes were less prevalent in Central and South American countries, these findings may be underestimations due to limited genomic data. Therefore, whole-genome sequence-based studies focusing on aminoglycoside resistance using a One Health approach in low- and middle-income countries should be encouraged.