AbstractBackgroundNeurofilament light chain (NfL) is a frequently used biomarker in humans for both diagnostic and therapeutic monitoring purposes in various neurologic diseases.Hypothesis/ObjectivesIt was hypothesized that dogs with diagnosed structural epilepsy (SE) would have a significantly higher serum NfL concentrations compared to dogs with idiopathic epilepsy (IE). The secondary hypothesis was that dogs would have a significantly higher serum NfL concentrations when measured within 7 days after a seizure compared to being seizure‐free for at least 30 days.AnimalsFifty client‐owned dogs presented to the neurology service for evaluation of seizures were enrolled. Fourteen dogs had SE and 36 dogs had IE.MethodsProspective cohort study performed on 52 serum samples obtained for NfL concentration measurement using single molecule array technology.ResultsThe median serum concentration of NfL in dogs with SE was significantly higher (109 pg/mL; range, 11.4‐741.3 pg/mL) than in dogs with IE (17.7 pg/mL; range, 5.8‐188 pg/mL; Wilcoxon rank sum test, P = .001). No significant relationship was found between serum NfL concentration and time of sampling in relation to the most recent seizure in dogs with IE.Conclusions and Clinical ImportanceSerum NfL may serve as an adjunctive biomarker for the differentiation of SE and IE.