Tight-head prop Adam Jones has been ruled out of Wales' clash with Australia in Cardiff on 3 December.
The 30-year-old's withdrawal leaves Wales light of prop options, as Paul James (thumb) is also out.
Craig Mitchell is unavailable for the Wales front-row, as the prop will not be released by English side Exeter.
Gethin Jenkins, Ryan Bevington, Scott Andrews and Rhodri Jones - barring any further injuries - will be the prop options against the Wallabies.
Wales have called uncapped Scarlets prop Jones into the squad to replace namesake Adam.
The 20-year-old can play at both tight-head or loose-head and has been earmarked as a possible contender for a place in Wales' squad for the 2015 World Cup.
Likewise, Andrews is also one for the future at international level, having been capped just once in the game against the Barbarians last June.
"Scott Andrews hasn't had a lot of rugby and we've got a lot of time for Scott Andrews," Gatland said.
"But we understand the pressure the regional coaches are under. It's about performance, it's about results and I just feel as a young tight-head he just needs to play.
"And he needs to have a couple of years playing regularly. Yes, he's going to learn a couple of lessons, and he's going to get a couple of hidings in a couple of games and I think in three or four years we could see an outstanding tight-head in someone like Scott Andrews.
"But it [strength in depth at tight-head] is a concern for us and we're just going to have to work hard in developing someone in that position and creating some depth there."
France-based contingent Mike Phillips, James Hook, Lee Byrne as well as previously overlooked Sale scrum-half Dwayne Peel, and his number eight Sale team-mate Andy Powell are also unavailable as the Test falls outside an official International Rugby Board window.
Jones lasted 63 minutes of the Ospreys' win over Biarritz on 12 November and he was then named in the starting line-up to face Treviso, but failed a late fitness test.
The British and Irish Lions prop joins a growing list of Welsh players already ruled out because of injury.
Those include lock Alun Wyn Jones (toe), who is out until around Christmas because of a dislocated toe suffered in training which requires surgery.
Wing George North, who suffered a hip injury in the Scarlets' Heineken Cup win at Northampton, has been included in Gatland's squad despite fitness doubts.
Other injury concerns in the squad include Jamie Roberts, Rhys Priestland, Leigh Halfpenny, Dan Lydiate, Adam Jones, Luke Charteris and Huw Bennett.
Charteris, who has a wrist injury, and Alun Wyn Jones were Gatland's preferred second-row pairing when Wales finished fourth at the World Cup in New Zealand.
Ryan Jones replaces his namesake at lock for Ospreys' European visit to Treviso on Saturday and the versatile 30-year-old is now favourite to partner Bradley Davies in the second row for the Australia showdown at the Millennium Stadium.
"Alun Wyn suffered a foot injury in training this week," Ospreys head physiotherapist Chris Towers said.
"As a result, he is going to require surgery to repair soft tissue damage, which will be carried out over the weekend.
"Once he starts his post-op rehab work we will be in a better position to predict recovery timelines."
Flanker Lydiate is also a fitness doubt for the World Cup third-place play-off repeat in Cardiff.
But Ospreys openside Justin Tipuric passed a fitness test for the game against Treviso.