Belgian blue (BB) cattle have an 11-bp deletion in myostatin that causes skeletal muscle hyperplasia and increased muscle mass, leading to a ‘double-muscled’ phenotype. Preliminary data suggest that this phenotype may be associated with breed-specific hematologic and biochemical values. Therefore, in this study, we sought to compare hematologic and serum biochemical parameters in healthy BB and Holstein Friesian (HF) cows and to propose breed-specific reference intervals for BB cows. Hematologic parameters, total protein, creatinine, creatine kinase (CK) and aspartate transaminase (AST) activities, albumin, and globulins were measured in 183 clinically healthy adult BB and HF cows. There were significant differences between BB and HF cows in 17 of 27 measured parameters. BB cows had significantly higher creatinine concentration and CK and AST activities (p < 0.001). RBCs, hemoglobin, hematocrit (p < 0.001), MCV and lymphocytes (p < 0.05) were also significantly higher in BB cows compared with HF cows. The average N/L ratio was greater than 1 in both breeds. These results suggest that BB and HF cows have significantly different clinically relevant hematologic and serum biochemical values, and, therefore, breed-specific reference intervals should be used.