"All 20,000 bees died within 48 hours," Amanda Fruci, publicist for the Royal Ontario Museum in Toronto, told AFP.
"The cause is being investigated but we know for sure that it wasn`t colony collapse syndrome because that involves bees leaving a hive and never coming back, and in this case they all died in the hive."
In normal times, bee communities naturally lose around five percent of their numbers.
But with the syndrome known as colony collapse disorder (CDD), a third, half -- sometimes even 90 percent or all -- of the insects can be wiped out.
In the United States, government figures released last year showed a 29 percent drop in beehives in 2009, coming on the heels of declines of 36 and 32 percent in 2008 and 2007.
Mysterious decimation of bee populations have also been reported in Europe, Japan and elsewhere in recent years, threatening agricultural crops that depend on the honey-making insects for pollination.
Thousands of visitors had viewed the bees at work in a specially designed glass hive in the Royal Ontario Museum`s popular hands-on biodiversity gallery over the past two years.
They were perfectly healthy until last week when they suddenly died.
The museum has already ruled out starvation or errors by staff as causes of death, but said poor ventilation, a parasite or too few worker bees to keep the comb warm over the winter may be to blame.