Thiamine (vitamin B₁) deficiency in Great Lakes salmonines has been linked to consumption of alewife Alosa pseudoharengus. Thiamine deficiency has been recognized as a possible impediment to lake trout Salvelinus namaycush recruitment in the Great Lakes and Atlantic salmon Salmo salar recruitment in the Finger Lakes and Baltic Sea. Alewife invaded Lake Champlain in 2003 which provided an opportunity to investigate changes in thiamine concentrations in salmonine predators during an alewife invasion. We monitored egg unphosphorylated and total thiamine concentrations in lake trout and Atlantic salmon in 2004 and 2007–2019, assessed whether concentrations were associated with mortality, and examined thiaminase activity in alewife. Total thiamine concentrations in lake trout and Atlantic salmon were significantly lower than in 2004 for seven of the ten collection years for lake trout and for nine of the 12 collection years for Atlantic salmon. Mortality and signs of thiamine deficiency were observed in laboratory-reared Atlantic salmon free embryos but not in lake trout. Average thiaminase activity in adult alewife declined from 5200 pmol/g/min in 2006 to 1500 pmol/g/min in 2012. Our results provide further evidence that a diet that includes alewife reduces egg thiamine concentrations in salmonines. This effect was observed within four years of the invasion of alewife.