Tip from SpeedBumpPony: If you can get used to Hyperspeed, that can help with the hit window on some of the notes once you get the chord movements down.
Strum the following combination in the Cheats sub-menu of the Options menu to activate the Hyperspeed trick:
(YO) (YO) (YO) (YO) (YO) (RY) (RY) (RY) (RY) (RB) (RB) (RB) (RB) (RB) (YB) (YO) (YO)
Original solution follows below:
This must be done in solo play, trying to achieve the score in co-op will not work.
Train Kept A Rollin' is one of the hardest songs in the game, so scoring 325,000 points is no easy feat.
This must be done on Expert, as the score is unattainable on any lower difficulty. There are 1400 notes on the Expert version of this song, and the number varies from about 93-96% of notes needing to be hit to achieve 325,000.
There's no real easy way to exploit this song for an easy achievement, it's just one of those cheevos that requires some good ol' fashioned elbow grease.
Go into Practice Mode and practice the song from the section TRAIN'S A COMIN' to CABOOSE. This is where the majority of your points will come from, as the section is filled with lots of chords. The first half mostly consists of difficult solos with verses that have very little point value unless you get the Star Power activation just right. While you may be good enough to get 130K in the first half of the song, you still have to hold it together to get the next 200K or so. The good news is a monster score like that is very attainable in the second half of the song.
The second half of the song is much easier than the first, provided that you are able to move your fingers quickly enough to hit some of the chord changes while not missing crucial Star Power phrases. If there's a section of notes you can never hit in the last half of the song, and there's a Star Power phrase in that section, blatantly miss the non-starred notes if it means getting that Star Power phrase that you would normally miss flailing through the section. ALWAYS activate on the chords, which will usually be the verses.
And of course, play the song over and over again, obviously. Most people sneak in on this achievement by the slightest of margins.
Another practice strategy thanks to Acceleretto: "I'm just doing Verse 3 (the start of those bastard chords) in Practice and going up a speed each time I FC that bit."