The story begins by introducing George, Harris, Jerome and Montmorency, a fox terrier. The men are spending an evening in J.'s room, smoking and discussing illnesses they fancy they suffer from. They conclude they are all suffering from 'overwork' and need a holiday. A stay in the country and a sea trip are both considered, then rejected after J. describes the bad experiences had by his brother-in-law and a friend on sea trips. The three decide on a boating holiday up the River Thames, from Kingston upon Thames to Oxford, during which they will camp, notwithstanding Jerome's anecdotes about previous experiences with tents and camping stoves.
They embark the following Saturday. George must go to work that morning ("George goes to sleep at a bank from ten to four each day, except Saturdays, when they wake him up and put him outside at two"), so J. and Harris make their way to Kingston by train. They are unable to find the correct train at Waterloo Station (the station's confusing layout was a well-known theme of Victorian comedy) so they bribe a train driver to take his train to Kingston, where they collect their hired boat and start the journey. They meet George up-river at Weybridge.
The remainder of the story describes their river journey and the incidents that occur. The book's original purpose as a guidebook is apparent as the narrator describes passing landmarks and villages such as Hampton Court Palace, Hampton Church, Monkey Island, Magna Carta Island and Marlow, and muses on historical associations of these places. However, he frequently digresses into humerous anecdotes that range from the unreliability of barometers for weather forecasting to the difficulties encountered when learning to play the Scottish bagpipe. The most frequent topics are river pastimes such as fishing and boating and the difficulties they present to the inexperienced and unwary.
