The province's Agriculture Department is trying to determine what happened at Barclay Brook, where thousands of fish died in 2011 and 2012 after pesticides from farmers' fields seeped into the water.
Government spokesman Wayne MacKinnon says pesticide run-off could be the cause of the latest fish kill, but water samples collected on the weekend have yet to be tested.
"That's probably a good supposition, although we don't know for sure," he said in an interview.
"Certainly, not enough effort has been made to contain run-off — if that in fact is what the case is here. We've expanded buffer zones and worked with producers in the area to identify some practices to be mitigated."
MacKinnon said the problem could be traced to the increased prevalence of severe weather. He said P.E.I. experienced heavy rainfall on Friday, the same day the fish kill was reported.
In all, about 100 dead fish were spotted in the brook, which flows into the Trout River near O'Leary.
MacKinnon said it's unlikely more dead fish will be spotted in the days to come.
"They get consumed fairly quickly or washed out to sea," he said.
Later Monday, the P.E.I. government said in an email that it was investigating a separate report of dead fish in Carruthers Brook, which runs into Mill River. The government could not be reached for comment.
In July 2012, about 2,000 dead fish were scooped out of a three-kilometre stretch of Barclay Brook near Coleman, and a much smaller fish kill was reported from the same area in 2011.
Environmentalists and sport fishing groups have been complaining about the problem for years.
"It's disappointing but quite predictable," said Peter Bevan-Baker, leader of the provincial Green party. "With the rain that we've had, all of the dots add up and point towards it being a pesticide problem."
Bevan-Baker said legislation requiring buffer zones between waterways and farm fields is not working, adding that the real problem is the excessive use of pesticides.
"We need to become less reliant on food that require poisonous chemicals in order to grow," he said.
"The heavy, sudden rainfalls and the unusual climatic things that are happening perhaps suggest that the measures that may have worked in the past are no longer up to the job."
The Sierra Club of Canada has said the use of pesticides on the Island should be reduced and that the province should better enforce buffer zone regulations.
In September 2011, P.E.I. farmer Avard Robert Smallman was fined $3,000 after he pleaded guilty to farming within 200 metres of a watercourse boundary without the protection of a grass headland.
The violation was discovered after Environment Department officers were called in following fish kills in the Trout River, Big Jacques River and Mill River. Environment Canada confirmed that it found traces of a pesticide in bottom sediment in the three rivers.
In July 2010, dead fish were discovered along a two-kilometre section of the Montrose River in West Prince area.
Three years earlier, fish kills were reported after heavy rain along the Dunk River and Tryon River. Investigators were never able to determine the cause, but several farmers were cited for violating P.E.I. environment rules.
— by Michael MacDonald in Halifax
Note to readers: This is a corrected story. A previous version incorrectly reported that O'Leary is in eastern P.E.I.