Penn State Behrend’s local population of spotted salamanders, Ambystoma maculatum, was divided into smaller sub-populations by the Bayfront Connector highway in the year 2000. Currently, it is unknown how this habitat fragmentation has affected gene flow between the sub-populations. The purpose of this study was to determine how the Bayfront Connector has affected the genetic structure of each sub-population through analysis of microsatellites isolated from tissue samples. The results analyzed allelic richness, heterozygosity, and F-statistics.