Wildlife crossing structures are widely assumed to increase road permeability for a variety of animals, however, because construction of these structures may span decades, "before" and "after" studies that compare pre- and post-construction periods are rare. Our study will compare the genetic connectivity of six Western Toad (Anaxyrus boreas) breeding locations in the I-90 Snoqualmie Pass East Project area in Washington State. These data will be augmented by the use of radiotracking of adults and mark-recapture studies in the same area. Genetic samples will be analyzed via microsatellite genotyping, for which a large number of primers are already available for this species. Sequence data will be analyzed to quantify relationships between populations and test the prediction that I-90 poses a significant barrier to Western Toad populations North and South of the freeway, as well as the prediction that the genetic distance of the breeding populations is positively correlated to geographic distance. The results of this research will serve as baseline data and may be used as a comparative tool for future studies that seek to determine the I-90 wildlife crossing structure effectiveness of promoting gene flow between Western Toad populations. This research may help identify threatened populations in the I-90 corridor and add to current knowledge about the home range of this species, as well as provide a framework for other research that seeks to apply a BACI approach to genetic connectivity studies involving crossing structures.