The Welshman currently occupies one of the automatic berths for qualification to the European team for the prestigious tournament at Gleneagles next month, but is yet to cement his place for certain.
In Prague on Thursday, the 38-year-old went round in a six-under 66, earning him a slender advantage over Sweden's Mikael Lundberg, who shot six birdies and a bogey in his five-under 67.
Donaldson benefited from a strong finish, picking up shots on the last two holes to end the day on top, while the leaderboard is congested behind Lundberg, with a cluster of 10 players all grouped on four under.
Stephen Gallacher and Joost Luiten, two of Donaldson's rivals for a place on Paul McGinley's Ryder Cup team, are further back in the field after both carded rounds of 70 to finish two under.
"It was a good day," said Donaldson, who knows a top-seven finish come Sunday would likely seal his spot on the team.
"I played nicely - the course was playing tough early on, quite windy and then died down late but that finish is pretty tough, so I'm happy with the result.
"I've got a bit of a cold but you just get on with it. I don't feel too bad considering but I'm here and playing nicely and more of the same.
"I know what I've got to do this week so it's just a case of going out and doing it. It's another golf tournament where you've got to go out there and play as well as you can and contend to win the tournament.
"I'm in a position to do that now; I just need more of the same over the next three rounds. You can't go too heavily into it, you have to go out there and it's just one shot at a time like any other week.
"There are other things circling around this result but you have to get on with it and keep doing the same things you've been doing every week."