Barriers along a watercourse and interconnections between drainage systems are dynamic landscape elements that are expected to play major roles in the dispersal and genetic structure of fish species. The objective of this study was to assess the role of these elements using creek chub (Semotilus atromaculatus) in the Mastigouche Wildlife Reserve (Québec, Canada) as model. Numerous impassable waterfalls and interconnections among drainage systems were inferred with geographic information systems and confirmed de visu. The analysis of 32 populations using seven nuclear microsatellites revealed the presence of three genetically distinct groups. Some groups were found upstream of impassable barriers and in adjacent portions of distinct drainage systems. Admixture among groups was also detected in some populations. Constraining phylogenetic procedures as well as Mantel correlation tests confirmed that the genetic structure is more likely to result from interconnections between the drainage systems than from the permanent network. This study indicates that landscape elements such as interconnections are of major importance for circumventing impassable barriers and colonizing lakes that are otherwise inaccessible. Such an approach could be relevant for determining the origins of fish species (i.e. native vs. introduced) in the context of conservation.