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River ecosystems are often fragmented by artificial structures, such as weirs. For anadromous species, these structures can impede access to upstream spawning sites and ultimately lead to severe population declines.This study focused on the freshwater spawning migration of the sea lamprey, Petromyzon marinus, an anadromous species threatened by habitat fragmentation across its native range. To quantify the cumulative impacts of multiple weirs on upstream‐migrating adults, and to explore the environmental factors affecting migratory movements, passive acoustic telemetry was applied to 56 individuals during their spawning migration in the heavily fragmented River Severn basin, UK.While 89% of tagged sea lamprey passed the first weir upstream of the release site on the main river, only 4% passed the fifth weir. For 85% of migrants, the upstream extent of migration was immediately downstream of a weir. Individuals that passed weirs upstream of the release site (n = 50) took 21.6 ± 2.8 days to reach their most upstream location, experiencing cumulative passage times at weirs of 15.7 ± 2.8 days; these delays constituted a median of 84% of total upstream movement times.Multistate models showed that the weir passage rates of sea lamprey in tidal and non‐tidal areas increased significantly when downstream river level and discharge were elevated. Upstream‐to‐downstream changes in direction were frequent downstream of weirs, but rare in unobstructed river sections.The results provided evidence for a cumulative effect of multiple weirs on sea lamprey movements, substantially delaying upstream migrants and limiting their spawning to atypical habitat. The results also demonstrated the crucial roles of high tides and elevated discharge events in enabling weir passage. Although the Severn Estuary features conservation designations for sea lamprey, this study reveals that barriers are inhibiting their upstream migration, a problem that should be addressed to assist sea lamprey conservation. [ABSTRACT FROM AUTHOR]