Hook was one of several players hoping to catch the eye before the visit of New Zealand and, although Gatland stated that the injury was just a knock, he could now miss the clash with the All Blacks.
Teenager Leigh Halfpenny was the shining light for Wales, scoring his first international try with a superb solo effort on the stroke of half time before sealing the win with Wales' fifth try in the final minute.
Halfpenny was joined on the scoreboard by Morgan Stoddart and two second-half penalty tries as Wales showed their forward power against a Canadian side ranked 15th in the world.
Gatland, who will act as forwards coach on the Lions tour of South Africa next summer, made 12 alterations from last Saturday's defeat to the Springboks, including handing scrum-half Martin Roberts a Test debut and centre Andrew Bishop given his first start.
Bishop had the chance to stake a claim for Ospreys colleague Gavin Henson's jersey after the 2005 Lions tourist was ruled out of the remaining Autumn Tests with an Achilles injury.
Wales went in search of a morale-boosting win over an inexperienced Canada side but fell behind in the ninth minute following James Pritchard's penalty from in front of the posts. Canada had their pride dented in the 55-0 hammering in Ireland last weekend, but the visitors came out with all guns blazing in a physical opening quarter.
Wales suffered a further blow with the loss of Hook who hoped to cement his place ahead of rival Stephen Jones ahead of their final Autumn Tests against southern hemisphere giants New Zealand and Australia.
But the dragons eased the early fears of the 59,326 crowd when Stoddart touched down to gave the hosts a 24th minute lead. Lock Ian Gough won a Canadian line-out and, after a sharp pass by Roberts, full-back Stoddart squeezed over in the left corner from Tom Shanklin's pass.
Canada responded eight minutes later and regained the lead when Pritchard fired over his second penalty from the half-way line.
The Cannucks nearly caused a major shock against Wales at last year's World Cup in France before the Welsh fitness and power proved too much. And it was a case of déjà vu at the Millennium Stadium as Wales hit back on the stroke of half-time through teenager Halfpenny's try. The 19-year-old Cardiff Blues wing, on only his second cap, skinned Justin Mensah-Coker before fending off Pritchard and Adam Kleeberger for a brilliant score.
Hook's replacement Dan Biggar extended Wales' lead to 13-6 in the 46th minute by adding his first international points with a routine penalty.
Wales then continued to pile on the pressure after the break and it eventually told just before the half mark as Stuart Dickinson awarded a penalty try. Then the Australian official was forced to give another touchdown in front of the posts on 71 minutes.
Ryan Smith scored a late consolation for Canada before Halfpenny had the final say by scoring under the posts in the last minute.
Wales: Tries - M Stoddart, L Halfpenny (2), penalty try (2), Cons - D Biggar (3) Pens - D Biggar
Canada: Try - R Smith Cons - J Pritchard Pens - J Pritchard (2)
Referee: Stuart Dickinson (Australia)