ObjectiveIndividual habitat preference can reduce intraspecific competition for resources and may differ between age groups, sexes, and adult phenotypes. The Channel Catfish Ictalurus punctatus is a widespread species occurring in diverse freshwater habitats. This species displays breeding philopatry, returning to nesting sites occupied in previous years. Larger Channel Catfish tend to nest in the main channels of large rivers, whereas smaller fish tend to prefer smaller tributaries. The purpose of our study was to determine whether this habitat segregation potentially associated with habitat preference affects the genetic structure of a population. We hypothesized that spatial segregation of breeding sites in the Ottawa River and its smaller tributaries at Lac des Chats reduced gene flow within the population, resulting in genetically differentiated demes associated with lacustrine‐like and fluvial habitats.MethodsMicrosatellite allelic data was collected from 162 Channel Catfish.ResultWe found little genetic variation between the Ottawa, Mississippi, and Madawaska rivers. Furthermore, our analyses suggested that the sampled specimens comprised one panmictic population. Fish from one site in the Ottawa River, however, were significantly differentiated from fish from a nearby site also in the Ottawa River as well as from fish from the Mississippi River tributary.ConclusionGiven that fish from sites further up the Ottawa River were not differentiated from fish from these sites, it is unlikely that geography can account for the differences observed; rather, assortative mating may explain the differentiation. We propose that panmixia within the population is caused by ontogenetic changes in habitat selection, straying individuals, or sex‐biased dispersal and philopatry.