Rosberg, unbeaten this season and seeking to become only the third driver to win the first five races of a season, lapped the sunny Circuit de Catalunya with a best time of one minute 23.078 seconds.
The only incident to trouble the German's continued domination, after setting the fastest time on Friday, was when his car went into what Mercedes called a protective 'limp home' mode early on.
The team detected a sensor problem, which they fixed.
Triple world champion Hamilton, 43 points adrift after four races, was 0.126 slower with the timings showing he was slightly slower than Rosberg in sector three.
The Briton had complained about the handling of his car on Friday, when he was only third fastest, and Mercedes made set up changes overnight.
Ferrari's Sebastian Vettel was a close third, 0.147 off the pace, with Dutch teenager Max Verstappen fourth fastest in only this third practice session for Red Bull after moving up from junior team Toro Rosso.
The 18-year-old had been third until Vettel popped in a late quick lap.
Verstappen's Australian team mate Daniel Ricciardo was fifth ahead of Ferrari's Kimi Raikkonen and the Williams of fellow-Finn Valtteri Bottas.
French driver Romain Grosjean, in the Haas, complained about Renault's Kevin Magnussen forcing him into the gravel after the Dane almost turned into him.
(Reporting by Alan Baldwin, editing by Sudipto Ganguly)
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