BACKGROUND Gentamicin is an aminoglycoside antimicrobial commonly used in horses at 6.6 mg/kg IV once daily. Therapeutic drug monitoring (TDM) can confirm desired peak concentration is reached for common bacterial isolates, and detect toxicosis associated with high trough values. OBJECTIVE Determine the relationship between gentamicin dose and plasma concentration in hospitalized horses, and identify a starting dose range to achieve peaks > 32 μg/mL. METHODS Sixty-five horses (2002-2010) receiving once-daily gentamicin with TDM performed (N = 99 sets). METHODS Retrospective study. Data from hospitalized horses including weight, dose, plasma peak, and trough gentamicin concentration, creatinine concentrations and presence of focal or systemic disease were collected from medical records. Peak concentrations measured 25-35 minutes after administration were included (N = 77). Data were divided into low (<7.7 mg/kg), medium (7.7-9.7 mg/kg) and high (>9.7 mg/kg) dose groups, and were grouped by the horse having focal or systemic disease. RESULTS Peak concentrations resulting from doses ≥7.7 mg/kg were 5.74 μg/mL (SE 2.1 μg/mL) greater than peaks from doses <7.7 mg/kg (P = .007). Peak concentrations was 3.6 times more likely to be >32 μg/mL if dose was ≥7.7 mg/kg (P = .04). There were no significant effects of dose on trough or creatinine concentration. At a given dose, horses with focal disease had higher peaks than those with systemic disease (P = .039). CONCLUSIONS These data suggest gentamicin dosage should be individually determined in horses using TDM, but support an initial once-daily dose of 7.7-9.7 mg/kg IV to achieve peaks >32 μg/mL and trough concentrations <2 μg/mL. Further studies evaluating the safety of doses >6.6 mg/kg are required.