In vitro cell models such as organoids are widely used in fundamental research and in translational medicine. In perspective, these models represent strong ethical (3Rs) and economic benefits for genotype-to-phenotype research in animal sciences. Organoids could be used to study how genetic variation affects key cellular processes underlying complex traits e.g. affecting health and disease resilience. To exploit this potential, efforts are still needed to assess to what extent organoids reflect the phenotypes of the original tissue they are derived from. As a first aim, we characterized the variability of pig intestinal organoids (enteroids) within different gut segments and/or animals vs. the original tissue. We generated organoids from the small intestine (duodenum, jejunum, ileum) and colon from 4 Large White commercial pigs at slaughter age using a protocol that is convenient for commercial slaughter conditions from cryopreserved biopsies of the gut as well as of other tissues. We compared gut organoids cultured in 3D and 2D to the original tissues in terms of morphology, number and growth rate, cell composition and barrier tissue integrity. Using transcriptomic profiling, we assessed the extent and fidelity of shared gene expression patterns to gain insight into the conservation of the molecular identity of the different gut segments organoids are derived from.