I'd have to check, but probably Mario Kart Wii. Monster Hunter Tri will probably pass it eventually, but I'm taking a break on it for now. I thought Smash Brawl would be a contender, but it's not even close and at this point I have little desire to go back. If I ever want to play a Wii fighter, it'll probably be Tatsunoko Vs. Capcom for the foreseeable future.
Twilight Princess, Radiant Dawn and Okami are all getting up there though, after multiple playthroughs for each. Also, I expect Metroid Prime Trilogy will get pretty high after I 100% all 3 games. Also, Shiren the Wanderer will probably get up there when I finally pull it off the pile'o shame and delve into it. I sunk nearly 100 hours into the DS game iirc.