The aim of this study was to assess the role of serum cardiac troponin I (cTnI), troponin T (cTnT), lactate dehydrogenase (LDH) and MB isoenzyme of CK (CK-MB) concentrations in the detection of cardiac injury in dogs with chronic mitral valve disease (CMVD). Fifty-five dogs with echocardiographic diagnosis of CMVD defined as cardiac thrill (Ct) and non-cardiac thrill (nCt) by auscultation-palpation, and ten control dogs were studied. Serum was extracted from a blood sample from each dog. Analysis of serum cTnI, cTnT LDH and CK-MB concentrations were performed. Mean +/- SD serum cTnI concentration was 1.9 +/- 0.38 ng/mL in group-Ct and 1.75 +/- 0.27 ng/mL in group-nCt, and cTnI concentrations in both the subgroups were significantly higher than in the control dogs (P < 0.01). cTnT and CK-MB concentrations were 0.031 +/- 0.02 ng/mL and 83.5 +/- 88.4 IU/L in group-Ct and 0.024 +/- 0.01 ng/mL and 51.6 +/- 47.4 IU/L in group-nCt, respectively. Mean cTnT and CK-MB concentrations in group-Ct were significantly higher than in control dogs (P < 0.01, P < 0.05). LDH concentration was 918 +/- 136.4 IU/L in group-Ct and 351 +/- 140.7 IU/L in group-nCt A significant difference in serum LDH concentrations was observed between the 3 studied groups (P < 0.05). In this study, compared with clinically healthy dogs, significant increases in serum cTnI, cTnT, LDH and CK-MB concentrations were detected in dogs with CMVD. The findings of this study indicate that the biochemical markers activity elevated with severity of the disease in dogs with CMVD. Measurement of cardiac troponins, as the newer markers than LDH and CK-MB concentrations, may be useful in the detection of cardiac injury and prognosis in dogs with CMVD.