This study was designed (i) to establish reference ranges for the essential trace element and background levels of toxic element exposure in the healthy/normal dog population, and (ii) to evaluate whether trace element concentrations vary in dogs suffering from different pathologies. Blood serum samples were collected from 187 healthy and diseased dogs at the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, University of Santiago de Compostela (northwest Spain). The samples were acid digested, and the concentrations of trace elements (Co, Cr, Cu, Fe, Mn, Mo, Ni, Se and Zn) and toxic elements (As, Cd, Hg and Pb) were determined by inductively coupled plasma-mass spectrometry (ICP-MS). This enabled us to establish reference ranges for the essential trace elements and the level of toxic element exposure in dogs, and to identify several clinical situations associated with variations in trace elements in serum. Relative to concentrations in healthy control dogs, statistically significant differences were observed in the concentrations of Cu (significantly higher in hepatic, inflammatory/infectious and oncological categories), Mo (significantly higher in renal category), Se (significantly lower in gastrointestinal category) and Zn (significantly lower in gastrointestinal, inflammatory/infectious and renal categories). Trace element concentrations can be a cause or consequence of disease, and the study findings indicate that trace element determination in serum provides useful information on the pathogenesis of certain diseases. Further research on the serum concentrations of trace elements, particularly in relation to other biochemical parameters and diagnostic tools, may provide valuable information for the diagnosis of diseases in dogs and the disease prognosis.