Guitarists Slash and Tom Morello actually did their own MoCap (Motion Capture) for their characters, so that the Slash and Tom in the game, have the same movements and styles as the real people.
The song Slither by Velvet Revolver appeared in commercials promoting the game, which was not in the actual game. This is the first (and to date, the only) instance of a Guitar Hero commercial featuring a song that isn't actually in the game (however it was eventually released as a download.)
First game in the series to feature boss battles.
First game in the series to get a release on a Nintendo system (the Wii.)
First game in the series to feature a bonus track that was a cover version and not a master recording (She Bangs the Drums by The Stone Roses).