Unravelling relationships between dispersal and population structure requires considering the impacts of assumption violations of indirect gene flow models in a given system. We combined temporal, individual and coalescent-based analyses of microsatellite DNA variation to explore the general hypothesis that unequal effective population size (Ne), asymmetric gene flow (m) and nonrandom (sex-biased) individual dispersal had an important effect on spatiotemporal population structuring in lake-dwelling brook charr (Salvelinus fontinalis). This integrative examination shed light on the dichotomous structuring observed between an outlet and three tributary-spawning populations and their potential for adaptive divergence. It revealed further that finer tributary population structuring incongruent with drainage structure has been shaped by asymmetric m from one population with a large Ne towards two populations of smaller Ne. Gene flow among the tributaries was also mediated mainly by male-biased dispersal. However, longer distance dispersal from tributaries to the outflow was female-biased. Spatially dependent sex-biased dispersal may have contributed therefore to gene flow at different levels of population structuring. Our results demonstrate how dispersal and population structure may interrelate to produce spatial variation in intraspecific diversity, and are therefore relevant for conservation programmes seeking to define conservation units or predict recolonization rates of extirpated populations.