BackgroundPlasma clearance of iohexol is a reliable method to assess the glomerular filtration rate (GFR). The relationship between aging and GFR in horses is unclear.Hypothesis/ObjectivesTo compare GFR estimated by iohexol clearance in horses of different ages.AnimalsTwenty‐one clinically healthy horses were enrolled.MethodsProspective study. Groups: (A) composed by 8 young horses (≤14 years old) with serum creatinine <1.5 mg/dL, (B) by 7 aged horses (≥20 years old) with serum creatinine <1.5 mg/dL, and (C) by 6 aged horses (≥20 years old) with serum creatinine ≥1.5 mg/dL. Iohexol was injected (75.5 mg/kg) through an IV catheter, and plasma samples were collected 5, 30, and 90 minutes later. Plasma clearance of iohexol was obtained by the high‐performance liquid chromatography‐ultraviolet method.ResultsThe GFR was 2., 2.1, and 1.45 mL/min/kg (median value) in Groups A, B, and C, respectively. Statistical analysis showed differences between Group A versus C for urea, Group A versus B and A versus C for creatinine, and A versus C for GFR.Conclusions and Clinical ImportanceGlomerular filtration rate was significantly reduced in aged horses with serum creatinine ≥1.5 mg/dL compared to young horses with creatinine <1.5 mg/dL; no differences were obtained between young and aged horses with creatinine <1.5 mg/dL. Glomerular filtration rate evaluation should be considered in aged horses even if the plasma creatinine values are normal.