The ever-rising prevalence of resistance to first-line antibiotics among clinical Escherichia coli isolates leads to worse clinical outcomes and higher health care costs, thereby creating a need to discover its basis so that effective interventions can be developed. We found that the H30 subset within E. coli sequence type 131 (ST131-H30) is currently, and has been since at least 2004, the main E. coli lineage contributing to key resistance phenotypes—including extended-spectrum-beta-lactamase (ESBL) production, fluoroquinolone resistance, multidrug resistance, and dual ESBL production-plus-fluoroquinolone resistance—at a United States tertiary care center with a rising prevalence of ESBL-producing E. coli isolates. This identifies ST131-H30 as a target for diagnostic tests and preventive measures designed to curb the emergence of multidrug-resistant E. coli isolates and/or to blunt its clinical impact.