Natural selection is presumed to be the driving force behind the occurrence of phenotypically and genetically divergent populations in sympatry within many north temperate freshwater fishes. If, however, these populations have different ancestral origins, history could also contribute to their divergence. We previously found evidence for the role of selection in the evolution of divergent outflow and inflow breeding populations of migratory brook charr (Salvelinus fontinalis) inhabiting postglacial Mistassini Lake (Québec, Canada). Here, we show that these populations do not have a common origin, through the use of admixture and spatial analyses with seven microsatellite loci. Divergent populations clustered into two different population groups when compared to samples from surrounding drainages, although inflow populations appeared to be more admixed between the two population groups than the outflow population. These results are noteworthy since outflow and inflow populations were monomorphic at mitochondrial DNA (338-bp sequence of the control region) and are only moderately differentiated (mean FST = 0.10). Colonization by two ancestral populations was also consistent with known outflow direction changes throughout lake formation. In addition to providing insight into how phenotypic divergence in sympatry may have been affected by the nature (i.e. timing and direction) of colonization of ancestral populations, our results also suggest that ancestral populations may have differed in their ability to colonize certain lake habitats.