Johnson was the highest-ranked casualty on day one of the WGC Match Play Championship on Wednesday.
Johnson, the world number six, was outplayed by Richard Sterne, with the South African claiming a 5-and-4 victory to oust the top seed from the Sam Snead bracket at Dove Mountain.
Sterne will next face 2012 champion Hunter Mahan, after the American dismissed Spain's Gonzalo Fernandez-Castano 3 and 2 in Marana, Arizona.
The Snead quarter also saw seeds two and six fall at the first hurdle, as Dustin Johnson dropped to a 4-and-3 loss to Sweden's Peter Hanson, while Keegan Bradley joined his American compatriots in exiting the event as another Swede Jonas Blixt beat him 2 and 1.
Also in that section, Japan's Hideki Matsuyama got the better of Germany's Martin Kaymer 2 and 1, and he will meet Northern Ireland's Graeme McDowell in the last 32.
McDowell was three down with three holes to play against American Gary Woodland, but won four straight holes to post a 19-hole victory.
Bubba Watson, fresh from his Northern Trust Open win on Sunday, continued his good form, with a 2-and-1 triumph over Finland's Mikko Ilonen, while France's Victor Dubuisson was a comfortable 5-and-4 victor over American Kevin Streelman.
Defending champion Matt Kuchar never trailed on his way to a 3-and-2 win over Austria's Bernd Wiesberger, as the second seed in the Gary Player bracket progressed to a match-up with compatriot Ryan Moore.
Moore edged a tight battle with Dutchman Joost Luiten 1-up.
Top seed in the Player section, England's Justin Rose endured a nervy finish to see off American Scott Piercy 1-up, and will meet South Africa's Ernie Els next after the latter saw off Scotland's Stephen Gallacher through 19 holes.
The only upset from the Player bracket was a big one, as England's Luke Donald – champion in 2011 – was thrashed 5 and 4 by Italian Matteo Manassero.
Manassero is now the only non top-eight seed in the quarter of the draw, and will next face American Jason Dufner, who edged countryman Scott Stallings after 19 holes.
Also, last year's PGA Tour Rookie of the Year Jordan Spieth beat Spain's Pablo Larrazabal 2-up, to book a last-32 date with Denmark's Thomas Bjorn, who beat Italy's Francesco Molinari 2 and 1.
Joining Donald on the scrapheap was countryman and 2010 champion Ian Poulter, who was beaten 2 and 1 by Rickie Fowler.
Poulter's exit was the most surprising of the Ben Hogan bracket, with Lee Westwood another English casualty – with American Harris English surpassing him 5 and 3.
English will face top seed Rory McIlroy, after the Northern Irishman progressed via a 3-and-2 victory over American Boo Weekley.
Charl Schwartzel and Jim Furyk will play off on Thursday, after respective wins over Kevin Stadler (3 and 2) and Chris Kirk (2 and 1).
Two-time winner this year on the PGA Tour, Jimmy Walker continued his resounding form with a 5-and-4 thrashing of Branden Grace, to book a clash with Fowler.
Spain's second seed Sergio Garcia endured a thrilling battle with Australian Marc Leishman, eventually winning after 22 holes.
Garcia next faces American Bill Haas, who ousted another Spaniard, Miguel Angel Jimenez, 4 and 3.
In the Bobby Jones bracket, third seed Steve Stricker did not survive the first round, as he was trumped by South Africa's George Coetzee 3 and 1.
Coetzee's next opponent, American Patrick Reed, also pulled off an upset as he beat Canada's Graham DeLaet 1-up.
Top seeds in the quarter Henrik Stenson (one) and Jason Day (two) both won, with the former seeing off a sturdy challenge from Thailand's Kiradech Aphibarnrat to salute 2 and 1.
Australia's Day saw off Denmark's Thorbjorn Olesen 2-up.
Fourth seed Brandt Snedeker edged England's David Lynn over 20 holes, and next faces Webb Simpson who defeated Thailand's Thongchai Jaidee 3 and 2.
Finally, honours for claiming the day's largest margin went the way of American Billy Horschel, who was on and off the course in 13 holes as he dispatched of Wales' Jamie Donaldson 6 and 5.