Migration is a fundamental aspect of the life history of many fish and must be well understood for targeted conservation and management. We used acoustic telemetry and otolith 87Sr/86Sr analysis, in conjunction with annual ageing, to study intraspecific variation in barramundi Lates calcarifer migration in the Northern Territory, Australia. Acoustic transmitters were implanted into 25 barramundi (420–1010-mm total length (TL); median 510 mm TL) from freshwater reaches of the South Alligator River and their movements tracked over >2 years. 87Sr/86Sr transect analysis was also conducted on otoliths of 67 barramundi from the Daly, Mary, South Alligator and Roper rivers. Acoustic telemetry showed that most fish remained in fresh water across wet and dry seasons. Higher rates of movement occurred during the wet season and a minority of fish moved into the estuary during high flows. Otolith chemistry analyses revealed high diversity in salinity histories among individuals. We integrated the telemetry and otolith chemistry data to examine migration as a function of the stage of sexual development, and have proposed a revised life history model that identifies three migratory contingents. We conclude that anthropogenic disturbance, including modified river hydrology, has the potential to alter the frequency of life history contingents in barramundi populations.