Pongo, a handsome Dalmatian owned by Roger, an artist, introduces his master to a charming young lady called Anita, who owns an equally charming lady Dalmatian, Perdita. Roger and Anita marry, and soon Perdita and Pongo are the proud parents of fifteen Dalmatian puppies. But Anita's chic and unscrupulous friend Cruella De Vil hires Jasper and Horace, two thugs, to kidnap the puppies and hide them in a remote country house. Here she already holds eighty-four puppies captive, intending to have a coat made from their skins.

Finding their human friends baffled by the crime, Pongo and Perdita appeal to the dogs of London. Signals sent out by the "Twilight Bark" are heard in Suffolk by a shaggy retired sheepdog, the Colonel, who with the help of Sergeant Tibbs locates the puppies and guides Pongo and Perdita to the spot. Soon all 101 Dalmatians are making their way back to London, closely pursued by Cruella and her minions. The dogs, disguised as Labradors, stow away on a lorry, and finally outdistance their enemies who end up foiled and furious. Roger and Anita decide to set up a home for all the Dalmatians.