Gene cassettes within class 1 integrons in bacterial chromosomes are crucial for spreading antibiotic resistance. This study investigated antibiotic resistance in Escherichia coli (E. coli) strains from the Rouge River, Saint Clair River, and Lake Saint Clair. We analyzed 66 E. coli strains for class 1 integrons with aminoglycoside resistance genes. Strains with the intI1 gene underwent polymerase chain reaction (PCR) amplification for class 1 integron variable regions, followed by sequencing with 3CS and 5CS primers. Of the 66 strains screened, 43 were found to contain Int1: Rouge River (23 strains), Lake Saint Clair (11 strains), and Saint Clair River (9 strains). Amplification, sequencing, and analysis of these Int-1 containing strains revealed the presence of 11 aminoglycoside gene cassettes. The aacA4 gene cassette exhibited resistance to multiple aminoglycosides, such as gentamicin, tobramycin, and streptomycin This broader resistance profile suggests that the aacA4 cassette may be linked to several resistance mechanisms, facilitated by horizontal gene transfer (HGT). On the other hand, certain cassettes when combined, such as “aadA1-aac(3) IV,” “aadA1-aacA4,” and “aadB-aacA4-aac(3) IV,” exhibit an even wider range of drug resistance, illustrating the complexity of antimicrobial resistance (AMR) mechanisms within these strains. For instance, the “aadA1-aac(3) IV” cassette provides resistance to essential antibiotics like streptomycin, rifampin, tobramycin, and nitrofurantoin, with some strains also resistant to ampicillin, ciprofloxacin, and ceftriaxone. Common resistance to rifampin and streptomycin across multiple configurations presents a significant treatment challenge. However, the lack of resistance to chloramphenicol and imipenem, and limited resistance to trimethoprim/sulfamethoxazole, suggests potential therapeutic vulnerabilities. These findings suggest that class 1 integrons are likely responsible for antibiotic resistance in the E. coli strains isolated from the three urban watersheds studied. Moreover, it was observed that the selection of specific gene cassettes within these integrons occurs over time, indicating a dynamic process where certain resistance genes are favored under selective pressures. This ongoing selection process underscores the adaptive nature of bacterial populations in urban water environments and highlights the critical role of class 1 integrons in the dissemination and persistence of antibiotic resistance.