Inflammatory periodontal disease (periodontitis) is widespread in dogs. This study aimed to evaluate site-specific changes in the canine gingival crevicular fluid (GCF) proteome during the longitudinal progression from very mild gingivitis to mild periodontitis. Periodontitis diagnosis in dogs requires anaesthesia, our ultimate aim was to develop a periodontitis diagnostic that could be applied to samples taken from conscious dogs. The objective of this work was to identify potential biomarkers of periodontal disease progression in the GCF of dogs.