About 1,000 fish, mostly trout, have been found dead in the Oona River near Dungannon, County Tyrone.
The Northern Ireland Environment Agency (NIEA) said pollution in the water is being classified as "high severity".
Investigating officers have identified a potential source of the discharge. Samples have been taken by the NIEA.
"We got a call on Easter Sunday at around four o'clock from a member of public to say they saw dead fish," Brian Luke from the NIEA said.
"We sent our officers to investigate and trout of all ages and sizes were found.
"So far, we see the pollution coming from an agriculture source in the area.
"This is an important fishing area. It's a devastating blow for the area.
"Investigations with scientists will be carried out. Biologists will be on site this Wednesday or Thursday.
"The pollution will eventually be washed away naturally. Normally that happens better in wetter conditions."
BBC NI reporter Gordon Adair has been to the scene.
"The Oona is one of those exceptionally pretty little rivers that are dotted around the country," he said.
"Thankfully in its finest part, the mature part of the river down near the village of Eglish, County Tyrone, it's fairly much unaffected.
"In fact, while I was there I saw a number of good healthy trout turning in the water, but it's in the upper reaches that it's a very different story.
"I'm led to understand that the environment agency have identified a probable source - obviously it's the early stage of the investigation - and that it is an agricultural source, slurry I am told, that somehow made its way into the river."
The environment agency will carry out a criminal investigation.
"The recovery of the river will be a matter for angling clubs who use it and for DARD (Department of Agriculture and Rural Development), they will have a look at how to repair the environmental damage that has been done and the possibility of restocking the river," the reporter added.
"Hopefully it will be given a good chance to recover.
"There is very little water in the river at the moment, so that lack of water has meant that the pollutant wasn't moved down stream with great velocity and hopefully that will have saved the lower reaches of it."