Objectives/Goals: Urinary tract infections (UTIs) cause significant morbidity, and many patients require multiple courses of antibiotics increasing the risk of antibiotic resistance. We determined the prevalence of urinary antibiotic heteroresistance (HR), which has been associated with treatment failures in vivo, to three first-line antibiotics for UTIs. Methods/Study Population: Clinical urine Escherichia coli isolates from patients in metropolitan Atlanta, Georgia in August 2023 were collected as part of public health surveillance performed by the CDC-funded, Georgia Emerging Infections Program (EIP). Only the first E. coli isolate collected for each patient was included in this study. Antibiotic susceptibility was determined through medical record review. HR to nitrofurantoin, trimethoprim-sulfamethoxazole, and fosfomycin was determined by population analysis profiling (PAP), where broth dilutions of E. coli were plated on increasing concentrations of the antibiotic. HR was defined as survival of >1 in 106 cfu but fewer than 50% survival at 1X antibiotic breakpoint (bp), resistant as > 50% survival at 1X bp and susceptible as survival of Results/Anticipated Results: Among 355 patients, 21 (5.9%) were resistant or intermediate to nitrofurantoin and 92 (26%) were resistant to trimethoprim-sulfamethoxazole. Antibiotic susceptibility data were missing from 5(1.4%) and 11(3%) of isolates for nitrofurantoin and trimethoprim-sulfamethoxazole, respectively. Susceptibility testing was not routinely performed nor reported for fosfomycin, thus excluded. PAP revealed that of the total 355 isolates, 3(0.84%) were heteroresistant to nitrofurantoin, 17(4.8%) were heteroresistant to trimethoprim-sulfamethoxazole, and 27(7.6%) were heteroresistant to fosfomycin. Of the isolates found to be susceptible by standard testing, 1(0.3%) and 9(3.6%) were heteroresistant to nitrofurantoin and trimethoprim-sulfamethoxazole by PAP, respectively. Discussion/Significance of Impact: Despite low rates of HR to nitrofurantoin and trimethoprim-sulfamethoxazole (0.84%, 4.8%), HR to fosfomycin was more frequent (7.6%). Given that susceptibility is not generally performed for fosfomycin, this could have implications for including fosfomycin as a first-line treatment for E. coli UTIs.