The 32-year-old American star was an oddsmakers favorite to collect the winner's green jacket this week at Augusta National after winning his past three starts.
Reigning US Open champion Johnson has not withdrawn from the year's first major championship and hopes to be fit enough to tee off in Thursday's final first-round group at 2:03 p.m. alongside compatriots Jimmy Walker and Bubba Watson.
"At roughly 3 p.m. today, Dustin took a serious fall on a staircase in his Augusta rental home," said his agent, David Winkle, in a statement. "He landed very hard on his lower back and is now resting, although quite uncomfortably.
"He has been advised to remain immobile and begin a regimen of anti-inflammatory medication and icing, with the hope of being able to play tomorrow."
But even if Johnson is able to start, his trademark long driving skills could be compromised, a potentially disastrous handicap over the 7,435-yard Augusta National layout.
The injury marks another setback in the checkered career of Johnson, who won his first major title last year at Oakmont.
Johnson withdrew from the 2012 Masters after a back injury suffered, his agent said, when he lifted as jet-ski.
At the 2010 PGA championship, Johnson carried a one-stroke lead into the final hole but received a two-stroke penalty for grounding his club in a bunker, doing so in a sandy area he did not recognize as a hazard.
Even in his major victory last June, Johnson had to play the final holes knowing he would likely receive a penalty over an earlier infraction after a rules official had told him to play on.
Johnson became the world number one by winning at Riviera in February and followed with World Golf Championships Mexico and Match Play titles last month, becoming the first player since 1976 to enter the Masters on a three-event win streak.
The last time a world number one missed a major was in 2015 when Rory McIlroy suffered an ankle injury a few weeks before he was to have defended his title at the British Open.
No world number one has won the Masters title since Tiger Woods in 2002 and no player since 1940 has made the Masters his fourth victory in a row.