The Satire of the Three Estates, or Ane Pleasant Satyre of the Thrie Estaitis, by Sir David Lindsay, has been revived by scholars and will be performed at Linlithgow Palace and Stirling Castle in June.
No texts survive of the original play but academics – including from Glasgow and Edinburgh universities – believe it will be the first performance of this once-lost version since 1540.
The shows recreate a production in Linlithgow that year, as well as a longer 1552 version staged in Cupar and at Edinburgh in 1554.
The Satire, often mooted as a play the National Theatre should stage, is an attack on the Three Estates in the Scottish Parliament – the clergy, the lords and Burghers, as represented by the characters Spiritualitie, Temporalitie and Merchant.
Professor Greg Walker, of Edinburgh University's School of Literatures, Languages and Cultures, said: "Just as Lyndsay, on the eve of the Reformation, sought to investigate Scottish national identity and promote social reform in his play, Scotland once more finds itself at a crossroads as it heads towards the independence referendum of 2014.
"People are again asking those questions raised in stark clarity in The Three Estates."