Intestinal epithelial organoids (IEOs) are an important tool for translational research, but many characterization studies have so far relied on gene expression analysis. Epigenetic mechanisms, such as DNA methylation, have key roles during intestinal development and homeostasis, but whether DNA methylation signatures in the intestines are cell-intrinsic or dependent on external cues was previously unknown. Now, using genome-wide DNA methylation profiling, Kraiczy et al. show that IEOs derived from fetal, paediatric or adult small and large bowel show stable gut segment-specific DNA methylation profiles that closely correspond to profiles from matched primary gut epithelium. Fetal IEOs showed changes in DNA methylation patterns during culture indicative of maturation that were partly regulated by TET1, a demethylating enzyme. Furthermore, distinct methylation differences were found in IEOs derived from a child with gastric heterotopia, highlighting the potential of this technology for disease-specific research models.