This paper presents in vivo mechanical characterization of the muscularis, submucosa, and mucosa of the porcine stomach wall under large deformation loading. This is particularly important for the development of gastrointestinal pathology-specific surgical intervention techniques. The study is based on testing the cardiac and fundic glandular stomach regions using a custom-developed compression ultrasound elastography system. Particular attention has been paid to elucidate the heterogeneity and anisotropy of tissue response. A Fung hyperelastic material model has been used to model the mechanical response of each tissue layer. A univariate analysis comparing the initial shear moduli of the three layers indicates that the muscularis (5.69 ± 4.06 kPa) is the stiffest followed by the submucosa (3.04 ± 3.32 kPa) and the mucosa (0.56 ± 0.28 kPa). The muscularis is found to be strongly distinguishable from the mucosa tissue in the cardiac and fundic regions based on a multivariate discriminant analysis. The cardiac muscularis is observed to be stiffer than the fundic muscularis tissue (shear moduli of 7.96 ± 3.82 kPa versus 3.42 ± 2.96 kPa), more anisotropic (anisotropic parameter of 2.21 ± 0.77 versus 1.41 ± 0.38), and strongly distinguishable from its fundic counterpart. The results are consistent with the tissue morphology and are in accordance with our previous ex vivo tissue study. Finally, a univariate comparison of the in vivo and ex vivo initial shear moduli for each layer shows that the muscularis and submucosa tissues are softer while in vivo, but the mucosa tissue is stiffer while in vivo. The results concerning the mechanical properties highlight the inhomogeneity and anisotropy of multilayer stomach tissue.