This study investigated the upstream spawning migration of anadromous river lamprey (Lampetra fluviatilis) in the River Trent, a heavily fragmented tributary of the Humber Estuary- the location of one of the largest river lamprey populations in the UK. River lamprey released upstream (n = 16) and downstream (n = 15) of Cromwell Weir were tracked from the upper estuary (Keadby) to upstream of the fourth weir encountered (Gunthorpe Weir). Receivers were also located in the Yorkshire Ouse catchment, which joins with the Trent at Trent Falls (to create the Humber Estuary). 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.