Dispersal, the movement of individuals from their natal population to their breeding population, has important ecological and evolutionary consequences. In lotic freshwater systems, landscape features such as barriers or connectors, can affect dispersal and thus gene flow. It is of special interest to characterize population genetic structure in the presence of impassable barriers because they can restrict dispersal and thus isolate populations. On the other hand, connectors such as man-made canals connect watersheds that would otherwise be isolated, thereby enhancing gene flow and potentially genetically homogenizing populations. Freshwater mussels (Order: Unionida), many of which are highly threatened, have a unique life history involving parasitism on host fish species for larval development and dispersal. Landscape features that affect fish passage will consequently affect mussel dispersal and the maintenance of populations and metapopulations. This manuscript explores how landscape features affect the population genetic structure and genetic diversity of the freshwater mussel species Lampsilis siliquoidea, Fatmucket, in the southwestern Lake Ontario drainage. Data from seven microsatellite loci revealed that 1) waterfalls act as barriers for gene flow between freshwater mussel populations within the same river, 2) populations above the barrier had lower genetic diversity, 3) there was no detectable effect of man-made canals on genetic diversity and, 4) populations that were not hydrologically connected by a canal were marginally more differentiated than the populations connected by a canal. Landscape features can alter the connectivity within and between drainages thus changing the scale at which management will be effective.