Increasing temperatures are allowing species globally to establish populations in previously inaccessible areas, potentially causing wide-ranging impacts on local ecosystems. Detecting natural dispersal barriers is therefore of enormous benefit to conservationists, who can prioritise areas more vulnerable to invasion. This is particularly useful in freshwater ecosystems, where there is often only one pathway between two given points. Here, we investigate the role of upstream slope and distance upstream as natural dispersal barriers to two fish species whose range in Scandinavia in shifting northwards, the northern pike (Esox lucius) and European perch (Perca fluviatilis). We used recolonisation of lakes that had previously contained both species before being treated with the piscicide rotenone to measure species ability to disperse past natural barriers. Slope parameters of connecting streams were obtained using land terrain maps. We tested both species ability to disperse past short steep increments and longer uphill stretches. We found that whilst E. lucius were limited by longer uphill stretches of river, P. fluvialtilis were limited by the distance between the lake and their source population. This will allow conservationists to prioritise efforts on lakes that are not protected by suitable downstream connections to source populations.