Abstract Background Mice, rats, and pigs are the three most used animal models when studying gastrointestinal peptide hormones; however their distribution from the duodenum to the distal colon has not been characterized systematically across mice, rats and pigs. We therefore performed a comparative distribution analysis of the tissue content of the major appetite- and glucose regulatory peptides: glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-1 (GLP-2), oxyntomodulin/glicentin, neurotensin, and peptide YY (PYY) from the duodenum to distal colon in mice (n = 9), rats (n = 9) and pigs (n = 8), using validated radioimmunoassays. Results GLP-1, GLP-2 and oxyntomodulin/glicentin show similar patterns of distribution within the respective species, but for rats and pigs the highest levels were found in the distal small intestine, whereas for the mouse the highest level was found in the distal colon. In rats and pigs, neurotensin was predominantly detected in mid and lower part of the small intestine, while the mouse showed the highest levels in the distal small intestine. In contrast, the distribution of GIP was restricted to the proximal small intestine in all three species. Most surprisingly, in the pig PYY was found in large amounts in the proximal part of the small intestine whereas both rats and mice had undetectable levels until the distal small intestine. Conclusions In summary, the distribution patterns of extractable GIP, GLP-1, GLP-2, oxyntomodulin/glicentin, neurotensin are preserved across species whereas PYY distribution showed marked differences.