Abstract The diversification of epigenetic regulation, including chromatin accessibility, is crucial for adaptation to diverse environments. Chromatin regulation in endocrine organs, such as the pituitary gland, plays a critical role in controlling various physiological processes underlying life-history traits, including growth, metabolism and reproduction. To understand the genetic architecture responsible for epigenomic divergence, we investigated chromatin accessibility in the pituitary gland using a parapatric pair of marine and stream ecotypes of the three-spined stickleback (Gasterosteus aculeatus). First, we characterized the distinct chromatin accessibility profiles of the ecotypes. Our chromatin quantitative trait loci (cQTL) mapping identified both cis- and trans-QTL across the genome. Further comparative analyses of effect size, direction of effect and functional enrichment suggest that cis- and trans-cQTLs may contribute differently to epigenomic divergence between ecotypes. These results suggest that epigenomic divergence could be a key regulatory mechanism underlying local adaptation and support the contribution of both cis- and trans-cQTL to the evolution of complex traits. This article is part of the theme issue ‘Ecological epigenetics at the intersection of behaviour and life history variation in non-model animals’.