Background: Genetic background can influence the expected values of hematologic and serum biochemical analytes in domestic animal species. Objective: The purpose of this study was to determine if there are breed-related differences in serum biochemical variables in healthy purebred dogs of 4 breeds and to develop appropriate reference intervals. Methods: Alaskan Malamutes (n=59), Siberian Huskies (n=78), Golden Retrievers (n=90), and English Setters (n=77) were included in the study. The dogs had a median age of 42 months (range 10-112 months) and each breed included a mix of intact and neutered dogs of both sexes. Serum biochemical profiles (Olympus AU400e) were performed along with physical examinations, CBCs, and urinalyses to ensure dogs were clinically healthy. Differences in the values of biochemical analytes were assessed nonparametrically and reference intervals for all breeds combined were calculated as the central 95% percentile. Results: Significant differences were observed between breeds for all serum biochemical analytes except alkaline phosphatase, glucose, and chloride. The analyte ranges had a large degree of overlap between the different breeds. Conclusions: Although many statistically significant breed-related differences in serum biochemical values were observed, the differences were small and unlikely to have clinical relevance or impact medical decision making.