The former South Africa international faced pressure to retain his job following a failure to win a trophy during his two seasons in charge.
The Bulls made the 2024/25 Sheffield Shield final, but lost a thrilling decider against South Australia.
Botha stepped into the job when former coach Wade Seccombe was asked to reapply for his position despite leading the Heat to an unlikely title in 2023/24.
Queensland Cricket on Tuesday confirmed Botha had quit.
"While we haven't achieved all of our on-field goals in that time, Johan has made a strong contribution towards the development of the next generation of Queensland and Brisbane Heat players," Queensland Cricket chief executive Terry Svenson said.
"Despite this decision, Johan's contributions to our high performance group have been valued.
"We wish he and his family every success in the future and thank him for his efforts across the Sheffield Shield, One Day Cup and BBL competitions."
Botha faced backlash last year when he said some players weren't doing enough to get back from injuries because they were still getting paid while absent.
"Players getting paid when they're injured, I don't think that helps Australian cricket," Botha said in October.
"If you play a game and you miss the next five (Sheffield) Shield games and then you are out for selection, you get paid all the way through."
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