We studied gene flow of non-native Brook Trout Salvelinus fontinalis in a 60-km section of continuous stream network in the upper Cache la Poudre River basin, where a large-scale reclamation effort to restore federally threatened Greenback Cutthroat Trout Oncorhynchus clarkii stomias (GBCT) is taking place. This effort—the Poudre Headwaters Project—represents the most important recovery effort of the state fish of Colorado and could result in a fivefold increase in total occupied GBCT habitat. However, the reclamation area is currently dominated by non-native Brook Trout; key steps to ensure the success of the Poudre Headwaters Project include removing non-native Brook Trout and physically isolating the area from reinvasion. We examined existing genetic population structure in the reclamation area to provide science-based guidance for effective Brook Trout removal and subsequent GBCT reintroduction. During 2018 and 2019, tissue samples were collected from 23 sites in the Long Draw region. We genotyped 792 Brook Trout using 12 microsatellite loci to examine genetic population structure within the reclamation area. Our results show that fine-scale genetic population structure is present among sites (F_ST = 0.038) and overall genetic diversity is comparable to previously published estimates throughout the native range of Brook Trout (H_O = 0.610; HE = 0.618). Analysis of genetic differentiation among sites indicates that sites in the reclamation area may function as a metapopulation, with fine-scale genetic structure present among tributaries that are connected by gene flow. Understanding how the landscape influences connectivity and population persistence will provide sciencebased guidance for Brook Trout removal efforts and allow managers to release GBCT in stretches of key habitat that ensure the highest probability of reintroduction success.