Serum thymidine kinase (sTK) activity was evaluated as a tumor marker for canine malignant lymphoma (ML). The objective was to investigate if sTK, as in humans, could be used as a prognostic marker for survival time in dogs with ML and if sTK could identify early signs of progression of disease in treated dogs. Serum samples from 52 dogs with ML were tested for initial TK activity. Samples from 21 normal dogs and 25 dogs with nonhematologic neoplasms were used for comparison. Forty-four dogs with ML were treated. Serum TK activity was measured in treated dogs before each treatment and every 4 weeks thereafter until relapse. Dogs with ML had 2–180 times higher TK activity (TK 5–900 U/L) than normal dogs (TK <7 U/L) based on the mean + 2 standard deviations. In the group of other neoplasms, only 2 dogs had a moderate increase (6.4 and 7.5 U/L) compared with the controls. Mean sTK activities in the dogs with ML that had gone into complete remission (CR) were not significantly different from activities in healthy controls (P= .68). Mean sTK at least 3 weeks before and at the time of relapse was significantly higher than activity measured at CR (P < .0001). Dogs with ML that initially had sTK >30 U/L had significantly shorter survival times (P < .0001). Furthermore, sTK activity reflected the clinical staging of ML. Measuring sTK can be used as a powerful objective tumor marker for prognosis and for predicting relapse before recurrence of clinically detectable disease in dogs with ML undergoing chemotherapy.