The wooden panelling, ceiling beams and fireplace from the Oak Parlour at Gwydir Castle, near Llanrwst, were sold to an American newspaper owner in 1921.
They were shipped to New York but have not been seen since the 1930s.
It is now hoped they can be retraced and brought back to Wales.
Judy Corbett, who owns Gwydir Castle with her husband, Peter Welford, said it was bought by William Randolph Hearst, an American newspaper tycoon in 1921.
The 16th Century linen-fold panelling, the fireplace overmantel and the carved and moulded ceiling beams were dismantled, crated up and sent to America.
"He ended up assembling it in his billiard room in his apartment in New York," she said.
The apartment was known as the Clarendon, said to be the largest apartment in the world.
It was partly demolished in the 1930s, but the couple know the panelled rooms were taken out and stored.
"This is where we lose track of it," they said.
"Where did our room go?
"Did the Hearst family hold on to it?  Was it sold? Was it donated to an American museum?
"We have spent years looking through archives and records but can find no trace of it anywhere."
Numerous items of furniture, along with another whole room, were also sold off in 1921.
The panelling from the dining room was also bought by Mr Hearst.
But it was later discovered in a warehouse belonging to the Metropolitan Museum in New York, and was brought back to Wales and reinstalled at Gwydir in 1996.
Ms Corbett added: "The story of the dining room shows that miracles can happen. We would love to find our second missing room."
The owners of a 16th Century Tudor mansion in Conwy county are appealing to people in the United States to help them find a missing room.