The 22-year-old, who broke the world record in both the heat and semi-final of the event on Tuesday, came agonisingly close to breaking it for a third time.
However, he again went sub 26s, coming home in 25.99.
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The gold was his fifth at the worlds, making him Great Britain's most decorated swimmer at the championships.
"I'm feeling very good. I'm so, so happy with my displays," Peaty told the BBC.
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“That felt really, really good even though I slightly messed up a stroke,” added Peaty.
“There’s definitely more in there somewhere but it will take a lot more hard work to make that happen. We’re only at the start of our four-year journey to Tokyo 2020.”
Joao Gomes Jr bested Cameron van der Burgh for silver.
The win means he has defended both titles he won last time out at the World Championships in Kazan in 2015.
Men's 50m Breaststroke Final results
1. Adam Peaty (Britain) 25.99
2. Joao Gomes Jr (Brazil) 26.52
3. Cameron van der Burgh (South Africa) 26.60
4. Felipe Lima (Brazil) 26.78
5. Kevin Cordes (U.S.) 26.80
6. Fabio Scozzoli (Italy) 26.91
7. Kirill Prigoda (Russia) 27.01
8. Ilya Shymanovich (Belarus) 27.27
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