AbstractBackgroundGreyhounds have been reported to have hyperhomocysteinemia (HHC), but the underlying mechanisms and clinical implications are unclear.HypothesisOur primary aim was to assess serum concentrations of homocysteine (HCy) and related analytes in Greyhounds and to identify a likely metabolic pathway for HHC. A secondary aim was to determine whether HHC is associated with evidence of oxidative stress.AnimalsHealthy pet Greyhounds (n = 31) and non‐sighthound control dogs (n = 15).MethodsAnalysis of serum HCy, cobalamin, folate, and methionine, and plasma cysteine, glutathione, and total 8‐isoprostane concentrations.ResultsHomocysteine concentrations were higher in Greyhounds (median, 25.0 μmol/L) compared to controls (13.9 μmol/L;P< .0001). Cobalamin concentrations were lower in Greyhounds (median, 416 ng/L) compared to controls (644 ng/L;P= .004) and were inversely correlated with HCy (r= −0.40,P= .004). Serum concentrations of folate, which is regenerated when HCy is converted to methionine, also were inversely correlated with HCy (r= −0.47,P= .002). Serum methionine concentrations were more than 4‐fold lower in Greyhounds (median, 3.2 μmol/L) compared to controls (median, 15.0 μmol/L), but this difference was not significant (P= .3). Plasma cysteine, glutathione, and 8‐isoprostane concentrations did not differ significantly between groups.Conclusions and Clinical ImportanceOur findings suggest a primary defect in conversion of HCy to methionine in Greyhounds, with related impaired folate generation. Ineffective cycling by methionine synthase could lead to secondary cobalamin depletion. Notably, low serum folate and cobalamin concentrations can be observed in Greyhounds without signs of intestinal disease.