ObjectiveTo compare plasma l‐lactate (LAC) values between samples collected from jugular and cephalic venipuncture in healthy horses and systemically ill horses.DesignProspective, experimental study.SettingLarge animal university teaching hospital.AnimalsThirty healthy adult university‐owned horses and 43 client‐owned horses presenting to the large animal hospital for elective surgical procedures or for emergent medical evaluation of systemic illness.InterventionsBlood samples were collected from the jugular vein (JV) and cephalic vein (CV) and placed in EDTA blood tubes prior to any medical therapy. LAC values were obtained with a handheld lactate meter at the time of blood collection.Measurements and Main ResultsLAC was higher in CV samples than JV samples in healthy horses (P < 0.001); however, all values were within the normal reference range. Similarly, LAC was higher in CV samples than JV samples in systemically ill horses (P < 0.001), but the median JV value was within normal reference range (1.9 mmol/L [17.1 mg/dL]), while the median CV value was outside the normal reference range (2.9 mmol/L [26.1 mg/dL]).ConclusionsThe CV is an alternative venipuncture site for assessing plasma LAC if the JV is not accessible or to preserve the JV for subsequent catheterization. However, in ill horses, the CV value may be outside the reference range when the corresponding JV value would have been within the reference range.