Champion-elect Hamilton trailed Max Verstappen by more than six tenths at Austin's Circuit of the Americas as the Dutchman set the pace in his Red Bull.
Ferrari's Sebastian Vettel came second in the final running before qualifying takes place later on Saturday, finishing 0.218 sec behind Verstappen, with McLaren's British rookie Lando Norris a surprise third.
Earlier, Charles Leclerc broke down following an engine failure. The young Monegasque was forced to pull off the course with smoke billowing from the back of his Ferrari.
Hamilton, fastest in Friday's second practice session, needs to finish only eighth to be certain on securing his sixth world title in Austin on Sunday.
The 34-year-old Briton has won on five of his seven visits to America. Qualifying takes place at 4pm local time (9pm UK) on Saturday.