Hailing the find as "sensational", the Austrian National Library (ONB) said bank employees had stumbled across the precious documents during a clear-out of the archives.
They had been there since 1926, according to the library.
"An unknown person deposited a leather-bound folder containing numerous personal documents, letters and photographs of the Vetsera family, including the farewell letters of Mary Vetsera from 1889," the ONB said in a statement.
The ONB said it hoped the discovery would help shed light on one of the world's great romances, which has inspired numerous movies, novels, ballets and plays.
The bodies of Mary Vetsera, two months shy of turning 18, and the 30-year-old crown prince were discovered in January 1889 at a hunting lodge in the Viennese woods near the town of Mayerling.
The prince is thought to have shot his lover before turning the gun on himself.
Their deaths sparked international headlines and became a source of speculation.
With the exception of a farewell letter from Rudolf to his wife, Stephanie, no other original documents related to the incident were believed to have survived.
"Until now it had been assumed that the letters had been destroyed after the mother's death," the ONB said.
Scientific research on the letters would begin in August, it added.
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