BackgroundOsseous‐associated cervical spondylomyelopathy (OA‐CSM) is a common condition of the cervical vertebral column that affects giant dog breeds. MicroRNAs (miRNAs) are small RNAs that regulate gene expression, and recent data suggest that circulating miRNAs present in biological fluids may serve as potential biomarkers for disease. The miRNA profiles of cerebrospinal fluid (CSF) from healthy dogs and dogs clinically affected by OA‐CSM have not been described.ObjectiveTo characterize the expression levels of miRNAs present in the CSF of normal Great Danes and identify differentially expressed miRNAs in the CSF of Great Danes clinically affected with OA‐CSM.AnimalsClient‐owned dogs: 12 control, 12 OA‐CSM affected.MethodsCerebrospinal fluid samples were collected prospectively. MicroRNA expression was evaluated using the NanoString nCounter platform and quantitative real‐time PCR.ResultsWe identified 8 miRNAs with significant differential expression. MiR‐299‐5p and miR‐765 had increased expression levels in the CSF of OA‐CSM‐affected dogs, whereas miR‐494, miR‐612, miR‐302‐d, miR‐4531, miR‐4455, and miR‐6721‐5p had decreased expression levels in OA‐CSM affected dogs compared to clinically normal dogs. Quantitative real‐time PCR was performed to validate the expression levels of 2 miRNAs (miR‐494 and miR‐612), and we found a 1.5‐fold increase in miR‐494 expression and a 1.2‐fold decrease in miR‐612 in the CSF of the OA‐CSM affected group (P = .41 and .89, respectively).Conclusions and Clinical ImportanceData generated from our study represent an initial characterization of the miRNA profile of normal canine CSF and suggest that a distinct CSF miRNA expression profile is associated with OA‐CSM.