Linear and heterogeneous habitat makes headwater stream networks an ideal ecosystem in which to test the influence of environmental factors on spatial genetic patterns of obligatory aquatic species. We investigated fine-scale population structure and influence of stream habitat on individual-level genetic differentiation in brook trout (Salvelinus fontinalis) by genotyping eight microsatellite loci in 740 individuals in two headwater channel networks (7.7 km and 4.4 km) in Connecticut, USA. A weak but statistically significant isolation-by-distance pattern was ubiquitous in both sites. In the field, many tagged individuals were recaptured in the same 50m-reaches within a single field season (summer to fall). One study site was characterized with a hierarchical population structure, where seasonal barriers (natural falls > 1.5 m in height) greatly reduced gene flow and weaker spatial patterns emerged due to the presence of tributaries, each with a group of genetically distinguishable individuals. Genetic differentiation increased when pairs of individuals were separated by high stream gradient (steep channel slope) or warm stream temperature in this site, although the evidence of their influence was equivocal. In a second site, evidence for genetic clusters was very weak at the most, but genetic differentiation between individuals was positively correlated with number of tributary confluences. We concluded that the movement of brook trout was limited in the study headwater streams, resulting in the fine-scale population structure (genetic clusters and clines) even at distances of a few kilometers, and gene flow was mitigated by “riverscape” variables, particularly by physical barriers, waterway distance (i.e., isolation-by-distance) and the presence of tributaries.