The mammalian stomach has conserved digestive functions but also shows major anatomical and physiological differences among species. However, broad comparative transcriptomic analyses across mammals and across ruminant stomach chambers remain limited. Here, we collected and integrated 111 bulk RNA-seq datasets from 11 mammalian species (humans, mice, monkeys, dogs, horses, donkeys, pigs, cattle, deer, goats, and sheep) and analyzed 12,048 shared one-to-one orthologous genes. We identified 56 conserved genes mainly involved in translation, ribosome biogenesis, protein homeostasis, and mitochondrial function. Pairwise comparisons revealed extensive transcriptional differences, with 2070–7110 differentially expressed genes across species pairs, together with distinct species-associated transcription-factor activity patterns. We also identified 25 ruminant-enriched genes associated with epithelial differentiation, ion transport, mucosal defense, and metabolism. Analysis of 69 samples from the rumen, reticulum, omasum, and abomasum of sheep, goats, cattle, and deer showed clear chamber-associated transcriptomic differences. The abomasum had a distinct expression program, with 235 enriched genes related to hormone regulation, secretion, transport, and digestion. Weighted gene co-expression network analysis further identified different gene programs in the forestomach and abomasum. Together, these findings reveal conserved, species-associated, and chamber-associated transcriptional features of the mammalian stomach and provide a useful framework for understanding its molecular diversity.