Natural capital accounting has the potential to yield important policy insights at multiple scales, but there remains a disconnect between regional-scale natural capital accounts and their use for informing policy. In this paper, we propose a roadmap that could lead to the creation of policy-relevant regional accounts, with steps split across an initial scoping phase and a subsequent development phase. We demonstrate the scoping steps in action with an application to the Colorado River Basin (“Basin”), a large watershed in the southwestern United States (U.S.) that has faced aridification and substantial high-profile tradeoffs around the use of its water and other natural resources. Drawing on prior U.S. Geological Survey science co-production efforts, we conducted a series of eight discussion sessions with 41 scientists and science representatives whose work is relevant to Basin water, riparian and riverine ecosystems, upland ecosystems and energy and minerals. We summarise participants' thoughts on key topics and economic linkages, their insights and questions of interest and their recommendations on existing scientific data sources and gaps. We evaluate the suitability of the available data for construction of System of Environmental-Economic Accounting (SEEA) Central Framework and SEEA Ecosystem Accounting accounts, including those for land, water, forests, energy and minerals and ecosystems (covering extent, condition and ecosystem services). We present a series of lessons learned during the scoping phase, as well as lessons that could be relevant for future practitioners engaging in the development phase. The information can help guide the development of timely and relevant regional-scale environmental-economic accounts in the U.S. and beyond.