ObjectivesTo compare serum concentrations of homocysteine in healthy dogs and those fitting the criteria for systemic inflammatory response syndrome and to compare these values to commonly measured B‐vitamins.MethodsStudy dogs were classified into non‐infectious systemic inflammatory response syndrome or sepsis groups and blood was drawn on Day 1 of the patient's hospitalisation for measurement of serum homocysteine, folate and cobalamin concentrations. Homocysteine concentration was measured in 51 clinically healthy dogs to serve as the control group.ResultsA statistically significant difference was found between the homocysteine concentrations of the healthy group when compared to non‐infectious systemic inflammatory response syndrome and sepsis groups. Homocysteine values were not correlated with folate, cobalamin or APPLEfast severity scores. Homocysteine concentrations were significantly lower in sick dogs when compared to the control group, which is dissimilar to the human population.Clinical SignificanceThe clinical significance of homocysteine changes in critically ill dogs is currently unknown.