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What's the guitar and model? When did this start to happen? How many cents sharp is it?

It's quite odd to have the 2nd and 4th frets in tune, but the 3rd fret out (barring a fret problem). Did you try a new string? Sometimes kinks can cause small problems in the bass strings.

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Less than 2 hours on the current string, Gibson songbird acoustic. It's like an 1/8th of a step sharp, (rough guess).

Could this be a problem with the neck?

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What method of checking your tuning are you using, and are you sure the 4th fret is in tune and not sharp as well?

Also, did you change to a heavier gauge set of strings?