Habitat fragmentation and degradation associated with suburbanization can have negative consequences on population persistence through the reduction of dispersal and concomitant gene flow. Using eight polymorphic microsatellite loci, we assessed the effects of forest fragmentation, water quality and hydroperiod on the genetic structure of a vernal pool-breeding amphibian, the wood frog (Lithobates sylvaticus), across 20 ponds in an unfragmented, forested landscape and 45 ponds in a landscape fragmented by moderate suburban development. Analyses were performed at the broad scale of the study area and at a fine scale, with spatially independent clusters of ponds selected within each landscape. Bayesian clustering approaches and AMOVA identified little population structure at the scale of the study area. At the fine scale, genetic structure was correlated with geographic distance and the presence of roads in two of the three fragmented clusters. Spatial autocorrelation analyses detected positive spatial genetic structure and restricted dispersal in one of the clusters in the fragmented landscape. We identified barriers associated with roads and suburban development in the fragmented landscape and with large bodies of water and elevation in the unfragmented landscape. Lastly, we found no biologically meaningful effects of water quality or hydroperiod on genetic variation. The results of this study indicate that wood frog populations are well connected, with high gene flow, across the landscape of southeastern New Hampshire, and that fragmenting features of suburbanization to date have a small but detectable impact on fine-scale genetic structure. The potential exists for greater impacts with higher levels of development or longer time scales. Our findings also highlight the importance of replication in landscape genetic studies, as the genetic response we detected varied with a gradient of fragmentation. © 2013 Springer Science+Business Media Dordrecht.