Van Der Burgh touched in a time of 26.77 seconds, a tenth outside the world record he set in Rome in 2009, with Australia's Christian Sprenger taking silver in 26.78 and Van Der Burgh's compatriot Giulio Zorzi bronze in 27.04.
It was the second gold of a fine session for South Africa after Chad le Clos won the men's 200 metres butterfly earlier on Wednesday.
Sprenger pipped Van Der Burgh to win the 100 metres breaststroke gold on Monday.
The 50 breaststroke does not feature in the Olympics.
(Reporting by Iain Rogers, editing by Pritha Sarkar)
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