Dead gulls found near Grovers Island, in the Clutha River near Roxburgh in Central Otago, will be tested to establish their cause of death.
Mykayla Robert, of Roxburgh, found the gulls while she was walking last weekend.
Posting on Facebook, Miss Robert commented that she was not sure how they died.
"I found at least 30-40 dead seagulls on that little island, mainly on the Clutha River side," Robert said.
Robert took pictures of the dead gulls.
"As you can see, there was one or two seagulls every metre right along the shore line," she said.
Department of Conservation (Doc) senior ranger, biodiversity, for Central Otago John Keene said a Doc ranger investigated the site that day and estimated up to 40 black-billed gulls were dead.
The ranger did not see any obvious signs on the carcasses of damage from animal predators, Mr Keene said.
The area was known for being a breeding colony site for black-billed gulls, an endangered species.
This was the second time mysterious deaths at the colony had been discovered, he said.
The Otago Daily Times reported in 2012 that at least 60 bird carcasses had been found.
The cause of death was a mystery and Jacob Dexter, who at the time was a Doc community relations ranger, sent some of the birds to Massey University for autopsies.
No traces of toxins or disease were found in the birds' systems.
The conclusion was "the cause of the mass die-off remains to be established".
The black billed gull/tarapuka is described by Doc as "the most threatened gull species in the world".
Their conservation status is "Threatened/Nationally Critical".
Keene said the cause of death for the 60 black-billed gulls found in 2012 remained a mystery, as Doc was not aware of the die-off until most of the carcasses were too decomposed for testing.
"There's a much better opportunity for valid testing with the current die-off, as the incident has been reported early when most of the carcasses are relatively fresh.
"While it's regrettable to lose any native birds, this particular loss of black-billed gulls is not expected to have a significant impact on the wider black-billed gull population," Keene said.
Five of the carcasses have been sent to Massey University for testing to determine the cause of death, he said.
Disturbing protected birds is an offence under the Wildlife Act 1953, and is punishable by imprisonment or a fine of up to $100,000.
Doc has asked that anyone who observes large numbers of sick or dying native birds should report it to their nearest Doc office or call Doc's emergency phone number 0800 DOC HOT (0800 362-468) or Ministry for Primary Industries pest and disease number 0800 80-99-66.