Objective: To describe and compare onset and intensity of thoracic duct (TD) coloration after injection of methylene blue into the diaphragmatic crus and mesenteric lymph node. Study Design: Experimental study. Animals: Adult dogs (n=18). Methods: Methylene blue (≤0.5mg/kg 1% solution) was injected into the left (n=9) or right (n=9) diaphragmatic crus via right 10th intercostal thoracotomy. TD coloration was graded over 10minutes. A right paracostal laparotomy was then performed in all dogs, and an equal volume of methylene blue injected into a mesenteric lymph node (n=18). TD color grading was repeated. Statistical analysis was performed on subject weight, volume of contrast agent injected between left and right crus, and number of successful outcomes between diaphragmatic crus injection and mesenteric lymph node injection. Results: TD coloration occurred in 6 dogs with left crus injection and 4 dogs with right crus injection with obvious staining present in 2 and 3 dogs, respectively. Successful outcome was noted in all dogs with mesenteric lymph node injection. The number of successful outcomes was significantly greater after mesenteric lymph node injection compared with diaphragmatic crus injection (P < .001). Conclusions: Methylene blue injected into the diaphragmatic crura and mesenteric lymph node was successful in coloring the TD; however, mean thoracic duct color grade and number of successful outcomes were significantly higher after mesenteric injection.