Globally, habitat fragmentation has had a major impact on the conservation and management of many species and is one of the primary causes of species extinction. Habitat fragmentation is loosely defined as a process in which a continuous habitat is reduced to smaller, disconnected patches as the result of habitat loss, restriction of migration or the construction of barriers to movement. Aquatic systems are particularly vulnerable to habitat fragmentation, and today an estimated 48% of rivers are fragmented worldwide. My dissertation evaluates how habitat fragmentation has influenced the populations of four different species of fish in the Lake Champlain basin. In chapter 1 I summarize the current state of habitat fragmentation research, I broadly describe habitat fragmentation, review how habitat fragmentation pertains to population genetics, and describe the legacy of habitat fragmentation in the Lake Champlain basin. In chapters 2, 3 and 4 I evaluate and discuss the impact of nine lake causeways on the population structure of slimy sculpin (Cottus cognatus), rainbow smelt (Osmerus mordax), and lake whitefish (Coregonus clupeaformis). The genetic effects of causeways are limited. However, causeways appear to have had a significant influence on rainbow smelt demographics, and the genetic structure observed in lake whitefish may be a product of reduced effective population size resulted from commercial harvest in the late 1800s. In chapter 5 I evaluate how the basin-wide population of tessellated darters (Etheostoma olmstedi) is naturally structured throughout Lake Champlain and three different major tributaries and evaluates the effect that different types of habitat fragmentation (dams, causeways, and natural fall lines) have on tessellated darter populations. Tessellated darters appear to be highly structured by river drainage but not by dams, causeways or fall lines. My dissertation highlights how comparative population genetic studies can be used to identify patterns of isolation within large populations. My results stress the value of reporting both the presence and absence of barrier induced population sub-structuring.