The shortgrass prairie of Colorado is the state’s largest and most diverse ecosystem, host to more than 200 plant and animal species across 27 million acres of land. A 1998 study by The Nature Conservancy showed that this ecosystem faced pressures from the development of Colorado’s highway infrastructure as well as private farm practices. While mitigation and land conservation efforts were already in place, analyses failed to achieve the scale necessary for a comprehensive evaluation that would improve the ecosystem. Marie Venner, from the Colorado Department of Transportation (CDOT), quickly recognized the opportunity for a conservation partnership that would incorporate disparate interests and rectify the technical deficiencies of piecemeal evaluations. This case study tells the story of CDOT’s partnership with the Federal Highway Administration, The Nature Conservancy, the United States Fish and Wildlife Service, and owners of the affected lands.