Lactate dehydrogenase (LDH) is commonly used in human cancer patients for prognostic purposes. Aim of this study was to determine the magnitude of serum LDH elevation in dogs with cancer compared with healthy dogs and dogs with non-neoplastic disease, and to verify whether it may support the diagnosis of specific malignancies. About 128 healthy dogs, 211 diseased dogs and 188 cancer dogs were enrolled. Dogs with cancer had significantly higher LDH than diseased (P < 0.001) and healthy dogs (P < 0.001), but large overlap was found. Dogs with lymphoma showed significantly higher LDH compared with dogs with carcinoma (P < 0.001) or mast cell tumour (MCT; P < 0.05) but not compared with other malignancies. When considering lymphoma and MCT, LDH levels were not different between early and advanced clinical stages. Measuring LDH levels may not be useful as a screening tool for cancer detection. More studies are needed to define its role in specific tumours.