Defending champion Marquez was chasing an historic 11th straight victory to start a campaign, but the Spaniard surprisingly came fourth - the first time he has failed to finish on the podium when he has completed a race.
That allowed compatriot and fellow Repsol Honda rider Pedrosa to claim a first race win since last October, the 28-year-old fending off Jorge Lorenzo (Movistar Yamaha) in a fascinating duel in Brno.
Pedrosa was elated with the win and also reserved special praise for his technical team.
"I didn't plan the race in this way, Jorge was so strong in the first two laps and opened it up immediately, so I had to change my plan and push the limits," he said at the post-race news conference.
"After the first laps I felt my bike was working well so I pushed hard and kept my rhythm until the end.
"Towards the end there was a little struggle with the rear grip and I was not able to go out strong in the corners.
"But I was able to hold the gap and win this race which makes me feel so special because it's been tough races [this season]. [The race] was special, I'm pleased for my team and to also see them very happy."