Gatland has warned his stars based in France and England that they will not be selected for his squad if they cannot secure full availability for the tournament.
He is preparing his side for their meeting with Australia at the Millennium Stadium on December 3, but the New Zealander is without France-based trio James Hook, Mike Phillips and Lee Byrne, plus Exeter prop Craig Mitchell and Sale duo Andy Powell and Dwayne Peel as the game falls outside the International Rugby Board's designated Test window.
That means clubs are not obligated to release their players for international duty and, although only scrum-half Phillips would have been a definite starter against the Wallabies, Gatland has made it clear that if players are not available for the start of Six Nations preparations, they risk finding themselves on the outside looking in.
Gatland said: "We knew the English and French-based players would not be available because this game is outside that IRB window.
"But we have said before, and we have been pretty consistent in this situation, that if those players in France and England can't get full release for the Six Nations then they will not be selected in the squad.
"We have been disappointed to hear that a couple of them had verbal agreements but it is not written in their contracts, which I sat down with them and told them to do.
"So the warning out there is that if you are going to leave Wales, make sure you have full release for all Wales games and squad sessions in your contract because if you don't you are not going to be selected, and it is a bit of a concern for us going forward."
Gatland also has a series of injury headaches to contend with prior to the re-match with Australia, who beat Wales 21-18 when the sides met in the third-place play-off at the World Cup.
Eight of Gatland's original 28-man squad joined the camp carrying problems, including George North (groin), Rhys Priestland (shoulder), Luke Charteris (wrist), Jamie Roberts (knee) and Dan Lydiate (ankle).
Gatland remains hopeful that his World Cup stars will recover in time, but one man who will not be available is Ospreys prop Adam Jones.
The formidable tighthead has suffered a recurrence of the calf problem that brought his World Cup semi-final to an early end, and has been replaced in the squad by uncapped 20-year-old Scarlets' forward Rhodri Jones.
With Mitchell unavailable and Paul James (thumb) another injury absentee, Gatland currently has Gethin Jenkins, Ryan Bevington and Scott Andrews as his other front-row options.
"Adam is not going to be available for us so we will replace him with another prop," Gatland said. "Scott Andrews has not played a lot of rugby and we have a lot of time for Scott.
"But we understand the pressure regional coaches are under, it's about performance and results but as a young tighthead he needs to play.
"He is going to learn a couple of lessons and have a couple of hidings in games, but in three or four years we could have another outstanding tighthead on our hands and we will need to work hard at creating some depth in that position."
There was some positive news for Gatland after assistant coach Rob Howley signed a new contract to remain as part of the national set-up until 2016.
The announcement follows last week's confirmation that Shaun Edwards had also agreed a new deal to take him through to the 2015 World Cup. Both Edwards and Howley will also work with Wales' age-grade sides.
Fellow backroom team members Neil Jenkins and Robin McBryde, who were WRU employees prior to Gatland's appointment in 2007, remain in place, with the latter's contract due to run out at the end of the season.
The notable omission from the squad was 104-cap fly-half Stephen Jones, who missed out with Ospreys number 10 Dan Biggar earning a recall.
But Gatland insists the decision does not mean the end of Jones' 13-year Test career, pointing out that the 33-year-old Scarlets' playmaker has not announced his retirement, unlike wing Shane Williams who steps down after the Wallabies' fixture.
"Stephen has not been selected but there are a couple of others (from the World Cup squad) not selected," he said.
"He hasn't retired, we know exactly what he offers us and this is a chance to give someone else an opportunity in the squad."
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