Disentangling ecological, behavioural and evolutionary factors responsible for the presence of stable population structure within wild populations has long been challenging to population geneticists. This study primarily aimed at decoding population structure of wild walleye (Sander vitreus) populations of Mistassini Lake (Québec, Canada) in order to define source populations to be used for the study of spatial partitioning using individual-based multilocus assignment methods, and decipher the dynamics of individual dispersal and resulting patterns of spatial resource partitioning and connectivity among populations. A second objective was to elucidate the relationships between biological characteristics (sex, size, age and population of origin) and an individual's probability to migrate and/or disperse. To do so, a total of 780 spawning individuals caught on five distinct spawning sites, and 1165 postspawning individuals, captured over two sampling seasons (2002–2003) were analysed by means of eight microsatellite loci. Four temporally stable walleye populations associated with distinct reproductive grounds were detected. These populations were differentially distributed among lake sectors during their feeding migration and their spatial distribution was stable over the two sampling seasons. Dispersing individuals were identified (n = 61); these revealed asymmetrical patterns of dispersal between populations, which was also confirmed by divergent admixture proportions. Regression models underlined population of origin as the only factor explaining differential dispersal of individuals among populations. An analysis of covariance (ancova) indicated that larger individuals tended to migrate from their river of origin further away in the lake relative to smaller fish. In summary, this study underlined the relevance of using individual-based assignment methods for deciphering dynamics of connectivity among wild populations, especially regarding behavioural mechanisms such as differential spatial partitioning and dispersal responsible for the maintenance of genetic population structure.