Background/Objetives: The bacterial ribosome is a key target for several classes of antibiotics, including aminoglycosides, macrolides, tetracyclines, and amphenicols. Although resistance to these antibiotics is well documented in clinical settings, ribosome-targeting antibiotic resistance genes have received comparatively little attention in studies comprising aquatic environments, where research has primarily focused on β-lactams and fluoroquinolones. Moreover, while plasmid-mediated dissemination of resistance is well recognized, the chromosomal integration of resistance genes in Escherichia coli remains underexplored. Methods: In this study, E. coli isolates were recovered from two contaminated aquatic environments in Panama: surface water from the Juan Díaz River and influent wastewater from the Panama City wastewater treatment plant. Results: Overall, 80.8% of the isolates exhibited resistance to aminoglycosides, 37.4% to tetracycline, and 18.2% to chloramphenicol. Resistance genes against these antibiotics were identified via PCR, and their genomic location (plasmid or chromosome) was determined by whole-genome sequencing. Our results revealed a higher prevalence of plasmid-associated resistance genes in river isolates, while chromosomal integration was more frequent among wastewater isolates. Notably, ribosome-targeting antibiotic resistance genes were more frequently detected than those conferring resistance to β-lactams, quinolones, and sulfonamides together. Conclusions: These findings highlight distinct mechanisms underlying the dissemination of ribosome-targeting antibiotic resistance genes in aquatic environments, where pollutant pressure in surface waters may favor plasmid maintenance, while chromosomal integration may represent a strategy to reduce the fitness cost associated with plasmid carriage and ensure stable resistance persistence.