Habitat corridor construction is an important technique for re-establishing connections between fragmented habitats. Yet, the effectiveness of habitat corridors to increase gene flow among fragmented populations is not well studied. In 2008 and 2009, a prairie habitat corridor was created in the Green River watershed of south central Kentucky, USA to protect water quality and encourage movement of native wildlife as part of the US Environmental Protection Agency Conservation Reserve Enhancement Program (CREP). In 2008, prior to the establishment of the habitat corridor, we documented the population genetic structure of 6 butterfly species (Chlosyne nycteis, Cupido comyntas, Phoebis sennae, Phyciodes tharos, Pterourus glaucus, and Pterourus troilus) with different habitat requirements and different expected responses to corridor construction using Randomly Amplified DNA Fingerprint (RAF) markers. STRUCTURE analysis of these markers subdivided each butterfly species into 2 to 8 subpopulations in the Green River watershed. By collecting data in the early stages of habitat restoration, we have established a baseline to compare with data obtained after the corridor has matured to determine the population genetic effects on previously isolated butterfly populations and to provide information about the conservation value of habitat corridors in general.