Gatland is awaiting results of a scan on Jamie Roberts' leg injury later today, while fellow backs George North, Manu Tuilagi and Tommy Bowe were also unavailable for selection.
Billy Twelvetrees, Brad Barritt, Christian Wade and Shane Williams will all start game six as a result, with the latter three still to arrive in Australia following their call ups in the early hours of this morning.
But rather than bemoan his bad luck, Gatland is confident his players will produce another display for him to be proud of and one which hands him some substantial selection headaches for next weekend.
"There's no way that we're going out there on Tuesday sacrificing a game. The motivation is there," said Gatland.
"That's what last night was about - going out and performing. I thought we performed extremely well and I'm sure those players that take the field on Tuesday will be incredibly motivated to continue the tour unbeaten.
"But it's not just that, there's still an opportunity for players on Tuesday to put their hand up and say 'I want to be a part of that Test XV or Test 23'.
"There's a lot of harmony and unity in the squad. Players go out on Tuesday with that chance to continue our unbeaten record but also to put themselves in the spotlight, too.
"It's important for the whole squad. It hasn't been about 15 or 23 players, it's been about the 37 or 41 or whatever it is now. That's been the whole focus.
"The players have been talking about (the fact that) it's your responsibility when you're wearing that jersey and someone hands over the baton. We've kept that momentum and it's been really pleasing and really positive how the players have done that and responded to that challenge.
"I'm sure on Tuesday night that those players who take the field will feel a massive responsibility to go out there and continue what we've been doing."
Gatland admits that producing a top performance against a side as well-versed and talented as the Brumbies with little training time together will be a big ask but he sees enough skill, spirit, familiarity and experience in his team to ensure that The Lions will head towards the first Test in a positive frame of mind.
"It's going to be tough. We're well aware that for the last two or three weeks the Brumbies have been lying in camp preparing for this game, waiting for us," added Gatland.
"Today was a recovery day, we travel tomorrow, we train tomorrow with a captain's run. We realise it's going to be tough on a few players, but then there's some combinations and experience that we looked at there with Billy and Brad knowing each other pretty well.
"Shane Williams will come in and, having worked with him for a long time, I think he'll fit pretty well into our game plan and understanding of what we're trying to do. There are some combinations there but also some experience in terms of players having been there and done that before and knowing what's expected of them.
"Everyone has to have the belief that you can go out and perform on Tuesday, put your hand up and get selected for next Saturday. Having this game before the first Test is ideal."