The objective of this study was to compare the effects of anticoagulant and no anticoagulant on routine biochemical analytes in domestic pigeons (Columba livia domestica). Blood samples were obtained from 8 clinically healthy pigeons. The sample obtained from each bird was divided into 4 blood collection tubes containing either ethylenediaminetetraacetic acid (EDTA), lithium heparin, sodium citrate, or no anticoagulant. The concentrations of creatinine, uric acid, triglyceride, total cholesterol, glucose, phosphorus, calcium, magnesium, total protein, albumin, and iron, and the activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were measured in blood from each of the blood collection tubes. The values of the measured parameters, with the exception of iron, were significantly lower in the citrated plasma samples compared with the serum samples, even after correcting for dilution. In the lithium heparin plasma samples, significant decreases in albumin, triglyceride, calcium, total cholesterol, and ALP, and a significant increase in iron, were observed compared with the values in the serum samples. The concentrations of total protein, creatinine, glucose, total cholesterol, triglyceride, magnesium, calcium, and phosphorus, as well as the activities for AST and ALP, were significantly lower in the EDTA plasma samples compared with the serum samples. In conclusion, the anticoagulants had significant effects on most of the measured parameters compared with serum. The findings of the present study suggest that a lithium heparin sample is the most appropriate plasma sample for the measurement of blood biochemical parameters in the domestic pigeon.