We studied current functional connectivity along a developing grassland corridor in the Midwestern U.S. and focused on connectivity for Greater Prairie-chickens (Tympanuchus cupido). Greater Prairie-chickens are an area-sensitive species that rely on grasslands and are an indicator species for the tallgrass prairie ecosystem in North America. The coordinated multi-agency Prairie Landscape Conservation Plan is one of the largest planned and implemented conservation corridors in the U.S., making this system ideal to examine factors associated with corridor functionality. We examined landscape functional connectivity and factors that impact genetic structure and gene flow of Greater Prairie-chickens to identify gaps in a planned prairie corridor that is currently being implemented. We collected feathers from 81 leks and obtained 31 hunter-harvested wings. We used a landscape genetic approach to evaluate eight hypotheses to explain functional connectivity and examine impacts of land cover and land use on isolation and genetic structure. Two populations were most supported based on 294 unique genotypes. Functional connectivity was influenced by land use, land cover, and anthropogenic development. Good connectivity exists within the Greater Prairie-chicken range, but improvements along the corridor in the northern portion of the extant range would be beneficial. We produced geographically specific recommendations as to where land management can potentially achieve the greatest impact. Our results help prioritize areas for ongoing conservation efforts, like prairie acquisition, restoration, and enhancement. Furthermore, identification of factors influencing connectivity in our study can be applied to improve conservation planning elsewhere in the tallgrass prairie ecosystem.