Abstract: Background - Vertical nystagmus can occur with disorders affecting the brainstem or cerebellum. The direction might correspond to underlying involved neuroanatomical structures. Hypothesis/Objectives - We hypothesized that downbeat nystagmus (DBN) would occur in dogs with cerebellar disease and upbeat nystagmus (UBN) would occur with brainstem disease. The objectives were to describe the prevalence of canine DBN and UBN, describe the sites of underlying MRI lesions and evaluate associations between MRI lesion location and DBN and UBN. Animals - 76 client-owned dogs with vertical nystagmus. Methods - Retrospective study. Medical records of dogs presenting with vertical nystagmus were reviewed and clinical findings recorded. MRI of the head were reviewed and lesions characterized. Descriptive and comparative statistics were performed using chi-square testing. Results - DBN was seen in 42/76 (55%) dogs and UBN in 34/76 (45%) dogs. All 6 dogs with a neuroanatomical localization exclusively to the cerebellum showed DBN. MRI lesions were multifocal in 35/76 (46%) and focal in 24/76 (32%) with 17/76 (22%) normal scans. Dogs with DBN had lesions located in the forebrain (21/42; 50%), midbrain/pons (18/42; 43%), medulla (20/42; 48%) and cerebellum (20/42; 48%). Dogs with UBN had lesions located in the forebrain (16/34; 47%), midbrain/pons (12/34; 35%), medulla (18/34; 53%) and cerebellum (11/34; 32%). No significant association was identified between any brain lesion location and direction of nystagmus (P>0.05). Conclusions and Clinical Importance - The direction of vertical nystagmus in dogs was not associated with MRI lesion location, however, DBN was associated with a cerebellar neuroanatomical localization.