Neonatal sepsis remains a major cause of morbidity and mortality, particularly in low-resource settings. Gentamicin is widely used for treatment; however, its narrow therapeutic index and variable neonatal pharmacokinetics increase the risk of accumulation and toxicity. Identifying factors associated with elevated gentamicin concentrations may support safer antibiotic use where therapeutic drug monitoring is unavailable. This study aimed to identify factors associated with elevated gentamicin trough concentrations among term neonates receiving once-daily intravenous gentamicin. This sub-study was nested within a prospective observational cohort evaluating the pharmacokinetics and safety of once-daily intravenous gentamicin at doses of 4.0 mg/kg and 5.0 mg/kg among term neonates with sepsis. The study was conducted from January to March 2025 in the neonatal unit of Mwananyamala Regional Referral Hospital (MRRH) in Dar es Salaam, Tanzania. A total of 250 neonates receiving gentamicin were included. Trough concentrations were measured at steady state, five minutes before the fourth dose. An elevated trough concentration was defined as more than 2.0 µg/mL. Factors associated with elevated trough concentrations were assessed using multivariable logistic regression, with adjusted odds ratios and 95% confidence intervals. Elevated trough concentrations occurred in 27.2% of neonates. Independent factors associated with elevated trough concentrations included age more than seven days (OR, 4.41; 95% CI, 2.16 - 8.99), weight less than 2.5 kg (OR, 2.10; 95% CI, 1.20 - 3.68), non-weight-based dosing (fixed-dose administration) (OR, 3.41; 95% CI, 1.45 - 8.05), vomiting (OR, 2.65; 95% CI, 1.22 - 5.74), diarrhea (OR, 8.26; 95% CI, 2.98 - 22.89), inability to suck (OR, 3.82; 95% CI, 1.26 - 11.56), and higher baseline serum creatinine (> 100 µmol/L) within the normal reference range (OR, 2.21; 95% CI, 1.03 - 4.73). More than one-quarter of neonates receiving once-daily gentamicin had elevated trough concentrations. Clinical and dosing-related factors may help identify neonates at risk of gentamicin accumulation and support safer antibiotic use where therapeutic drug monitoring is unavailable.