The 33-year-old Ukrainian improved to 53-3 and scored his 48th KO. Chambers lost for the second time in 37 fights.
Klitschko caught Chambers with a big right that nearly floored him late in the second round. Chambers staggered but survived the round by clinging onto Klitschko.
Otherwise, Klitschko jabbed his way through a largely dull fight, with Chambers mainly trying to limit the damage.
Chambers, known as "Fast Eddie," gave away 5 inches in height and 35 pounds in weight to the champion and his game plan was to get in close and try to connect inside. But Klitschko kept him away with his bigger reach and the effective left jab.
"I have to give him credit, he was (very) fast," Klitschko said. "He doesn't have much punching power and his game plan was gone by Round 6.
Before getting hit in the second, Chambers threw Klitchko to the canvas with a wrestling move, and once ended up carrying the Ukrainian on his shoulders after bending down twice.
"I don't think he ever really hit me, but it wasn't easy. I wanted to be too perfect maybe," Klitschko said.
Klitschko waited for his moment in the third, but Chambers mostly stayed away with arms raised. In the fourth, Klitschko tried to land some rights but mostly kept Chambers at bay with the left jab.
"When I was able to get past his jab, I couldn't land any combinations. He worked well with his jab," Chambers said later, wearing dark glasses.
Chambers landed a right hook to the side of Klitschko's head in the fifth but the champion finished the round strongly, getting through with a couple of rights.
With little action in the sixth, Klitschko easily dominated the seventh, repeatedly hitting Chambers with left jabs and following through with rights to the head, but Chambers did not appear in great trouble.
Klitschko said before the fight that he was in the best shape of his life, but his timing seemed slightly off in his first bout in nearly 10 months.
Klitschko hit Chambers with a short right late in the eighth but did not follow up.
Chambers had to change his damaged right glove at the start of the 10th, leading to a delay.
Klitschko's corner, including his older brother Vitali, the WBC heavyweight champion, urged him to step up the pace and knock out the American, but Klitschko kept his distance and relied on the jab to collect points.
Klitschko clearly tried to deliver the knockout punch in the final round and after chasing Chambers around the ring, he caught the American with a looping left to the side of the head that put Chambers down.
