Anthropogenic structures extensively fragment riverine systems, reducing longitudinal connectivity, inhibiting migration and leading to severe declines in many fish populations, especially for diadromous species. This study investigated the upstream spawning migration of anadromous river lamprey (Lampetra fluviatilis) in a heavily fragmented tributary of the Humber Estuary, the location of one of the largest UK river lamprey populations. Overall, this study quantified river lamprey migration, spawning habitat distribution and historic river levels to develop a novel empirical index to understand the impact of man-made barriers and prioritise their remediation. Passage at all weirs only occurred during episodic high river levels, often after prolonged delays with no lamprey passing below average levels for the time of year or utilising the fish pass at the first weir (T1) at the tidal limit. Barrier passage opportunities at the first four weirs were only possible for 30.3%, 38.7%, 52.1% and 6.7% of the migration period, but were lower and severely limited in 15 of the last 21 years. In addition, more lamprey (60%, n = 18) were last detected in reaches with no spawning habitat than in spawning habitat (40%, n = 12). Given the impassibility of, and lack of retreat from, T1 to other Humber tributaries, the River Trent is currently considered an ecological trap for a large proportion of lamprey that enter from the Humber Estuary. This passage should be urgently remediated, per the prioritisation index presented here, to aid river lamprey conservation, especially given their status as a designated feature of the Humber SAC.