This was Klitschko's eighth defence of his IBF title, the fourth defence of the WBO title he won against Sultan Ibragimov in New York in February 2008 and he was rarely troubled by the 27-year-old American.
Although Klitschko, 33, easily out-classed Chambers, ex-world champ George Foreman last week questioned how good the Ukrainian's opponents are and the champion failed to knock his opponent out until the final round.
"I must give Chambers plenty of respect, he was very quick," said Klitschko.
"I was looking for the knockout from the sixth round, but it didn't come until much later, so respect to him for that."
Just five seconds before the end of the fight, a huge left dropped Chambers to the canvas and gave Klitschko his 48th knockout and the 54th win of his career, with just three defeats.
But this was not an impressive performance.
Chambers, who suffered the second defeat of his career, came out with some strange tactics and kept looking to lift Klitschko off his feet, which he did successfully in the second round when he put the champion on the canvas.
The Ukrainian responded by pinning the American to the ropes with a flurry of punches and Chambers stumbled briefly before repeating his lifting trick to carry himself out of trouble.
With a 15 centimetre height advantage, Klitschko kept lining up the challenger and jabbed with his left throughout the third and fourth rounds, but Chambers used his agility to turn Klitschko and rarely allowed himself to be hit early on.
By the fifth, Chambers started looking to connect with Klitschko's jaw to test how well the champion can take a decent punch, but the American found no way through his opponent's guard.
Chambers came out for the sixth with his face showing signs of the punches Klitschko was landing through the Americans guard and by the seventh, the champion was clearly ahead.
Having landed some heavy shots to the head in the eighth, it was just a matter of time before Klitschko retained his title.
The challenger rallied briefly in the ninth, but Klitschko's superior fitness meant the champion was untroubled by the late effort and only a lucky punch was going to give Chambers victory.
The American had to change the glove on his right hand at the start of the tenth round which drew jeers from the crowd at the Esprit Arena, before Klitschko then landed two more big punches.
With time running out, the knockout punch came, but Klitschko certainly took his time before exerting his authority and will need to meet Britain's WBA heavyweight champion David Haye in the ring to truly dominate the division.