Seeking his third European Tour victory, the Welshman produced two straight birdies on his last two holes to top the leaderboard. He made a total of eight birdies and two bogeys at the Albatross Golf course near Prague.
''I played nicely all the way, really,'' Donaldson said. ''I just need more the same over the next three rounds. There's a lot of difficult holes.''
Sweden's Mikael Lundberg was one stroke back in second place, with a group of 10 players carding 68.
Donaldson currently has a qualifying spot for his debut Ryder Cup but still needs a good result to guarantee a place on Paul McGinley's team for next month's match against the United States at Gleneagles.
Play was delayed by an hour because of lightning.