Objective To investigate the antimicrobial resistance patterns of Escherichia coli isolates from infants younger than 90 days and analyze their relationships with age, infection site, and isolation time. Methods A retrospective study was performed by collecting Escherichia coli strains isolated from hospitalized infants aged ≤90 days in the neonatal ward of Capital Center for Children's Health, Capital Medical University from June 2020 to December 2023. Antimicrobial susceptibility testing was conducted. Relevant clinical information including age, infection site, and isolation time was collected to characterize the antimicrobial resistance of Escherichia coli from multiple perspectives. Results A total of 384 infants were included, among whom 253(65.9%) were in the neonatal group (age ≤28 days) and 131(34.1%) in the young infant group (29-90 days). The overall resistance rate of Escherichia coli was 72.4%, with a multidrug resistance rate of 21.0%. The neonatal group was more susceptible to invasive infections, whereas the young infant group exhibited higher antimicrobial resistance rates, with statistically significant differences observed in resistance rates to multiple antibiotics (P<0.05). Intestinal isolates showed higher resistance rates to β‑lactams, tetracyclines, and quinolones compared to the overall average, but lower resistance to sulfonamides. Blood isolates demonstrated higher resistance to β‑lactams. An increasing trend was observed in resistance to quinolones and tetracyclines over time. Among infants with multi-site infections, 72.9% had isolates with consistent antimicrobial susceptibility patterns, indicating clonal homology. Conclusions Escherichia coli isolates from infants aged 29-90 days exhibit relatively high antimicrobial resistance rates. In infants younger than 90 days, resistance patterns differ by age group and infection site, highlighting the need for rational antibiotic selection based on these factors.