The story of The Boys from Syracuse follows the twin brothers Antipholus of Syracuse and Antipholus of Ephesus, who are separated at sea when they are children. Years later, Antipholus of Syracuse arrives in Ephesus, where he is mistaken for his twin brother. This leads to a series of mistaken identities and comic complications.

The musical is based on William Shakespeare's play The Comedy of Errors, which itself was loosely based on a Roman play, The Menaechmi, or the Twin Brothers, by Plautus.