A chemical, thought to be caustic soda, is understood to have entered the water at Knockando burn in Moray in recent days.
The Scottish Environment Protection Agency (SEPA) confirmed it is investigating a potential pollution incident on the burn.
Fly fishing anglers say the salmon population at the burn has been wiped out and could take five years to recover, with a 2km stretch of the watercourse “destroyed”.
Duncan Ferguson, director of Spey Fishery Board, added that the pollution incident was one of the largest he had seen in 36 years of working on the river.
He said: “It’s a tragic event. It’s a really bad outcome and it didn’t have to happen.”
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Mr Ferguson said the chemical is thought to be industrially linked.
The incident comes during the peak period for fly fishing on the Spey, and just 10 days after another pollution incident at another Spey tributary.
Earlier, a number of salmon died after white paint was spilled into the Burn of Carron at Aberlour.
A spokesperson for SEPA said: “SEPA are investigating a potential pollution incident in a tributary of the River Spey and are working to identify the source and impacts.”
