Extended interval gentamicin dosing in neonates <35 weeks corrected gestational age (CGA) has been shown to achieve optimal serum peak concentrations compared to traditional once daily doses. However recommendations on exact dose and frequency vary. After a literature review, a dose of 5 mg/kg/dose every 48 hours was implemented at our institutions. To characterize concentration-time profiles achieved with a gentamicin dose of 5 mg/kg every 48 hours in neonates <35 weeks CGA. To identify gentamicin pharmacokinetic parameters in neonates <35 weeks CGA: half-life (t1/2), clearance, and volume of distribution (V1). Pharmacokinetics were prospectively analyzed after implementation of 5 mg/kg gentamicin as a single empiric dose for sepsis in neonates <35 weeks CGA (n=54). Serum samples were drawn at approximately 1 h, 24 h, and 48 h post dose. Target serum concentrations were 6–12 mg/L and 0.5–2 mg/L at 1 h and 48 h, respectively. Pharmacokinetic analysis was performed on available samples to determine if birth weight, gender, gestational age at birth, CGA, or postnatal age (PNA) affect gentamicin clearance, t1/2, V1, or serum levels. A one-compartment model best represented the pharmacokinetics of gentamicin for our population. Average pharmacokinetic parameters were: clearance 42±10 mL/kg/h, V1 0.578±0.087 L/kg, and t1/2 9.98±1.84 h. In all treatment courses when at least two serum levels were drawn (n=44), mean levels extrapolated to 1 h, 24 h, and 48 h were 8.24±1 mg/L, 1.6±0.48 mg/L, and 0.31±0.16 mg/L respectively. Neonates <14 days PNA had significantly higher gentamicin levels at 24 h (1.7±0.3 mg/L vs. 1.1±0.7 mg/L, P=0.002) and at 48 h post dose (0.34±0.14 mg/L vs. 0.2±0.2 mg/L, P=0.03). Clearance was significantly lower in neonates <14 days PNA (mean 38±5 mL/kg/h vs. 55±18 mL/kg/h, P=0.01) but was not affected by other covariates. V1 was not influenced by any covariates. A gentamicin dose of 5 mg/kg in neonates <35 weeks CGA and <14 days PNA results in post serum levels of 6–12 mg/L. Gentamicin clearance in neonates <35 weeks CGA was not affected by gestational age at birth, CGA, gender, or birth weight but increased as PNA increased suggesting the need to dose gentamicin more frequently than every 48 hours in neonates 14 days PNA. The risk of medication error may be reduced from this simplified approach.