The objectives of this study were (1) to measure normal thickness values of the muscularis, submucosal, mucosal and serosal layers in each gastrointestinal (GI) segment (gastric fundus, body and pyloric antrum; duodenum; jejunum; ileum; colon), and (2) to calculate a ratio of muscularis and mucosal layer thickness to aortic diameter measured at the level of the celiac artery (Musc:Ao and Muc:Ao) in each GI segment in a sample of healthy cats. Ultrasonographic examination of the GI tract was performed, and measurements of the individual layers in each GI segment were obtained in 38 healthy cats without clinical evidence of disease. The muscularis layer was significantly thickest in the ileum, compared with other segments, and it was thicker than the submucosa in all segments except the colon. The mucosa was the thickest layer in all segments, and was thickest in the duodenum and ileum. Measurements of the submucosal and serosal layers were not significantly different between all segments. Musc:Ao and Muc:Ao in each segment were 0.12 and 0.25 (gastric fundus), 0.12 and 0.18 (gastric body), 0.11 and 0.16 (pyloric antrum), 0.08 and 0.27 (duodenum), 0.08 and 0.22 (jejunum), 0.14 and 0.25 (ileum), and 0.05 and 0.08 (colon), respectively. Musc:Ao and Muc:Ao are clinically relevant values that can be used to objectively identify thickening of the muscularis and mucosal layers in response to GI diseases.