More than 20 years after the death of the Grateful Dead founder, the auction of the custom-made guitar fetched a total of $3.2m (Â£2.5m).
It was bought at a New York charity concert by Brian Halligan, CEO of marketing group HubSpot and a lifelong fan of the band - or a "Deadhead".
The money raised will go to the Southern Poverty Law Centre.
Halligan bought Wolf for $1.9m, including the buyer's premium, and had his $1.6m pre-premium bid matched by an anonymous charity.
Garcia was the best-known member of the Grateful Dead and was known for his guitar sound.
Devoted Deadheads identify five classic guitars which Garcia played at different times: Alligator, Rosebud, Lightning Bolt, Tiger and Wolf. The last two were auctioned together in 2002 and made more than $1.5m (Â£1.2m).
Wolf was put up for auction again on Wednesday night to raise funds for the advocacy group.
It got its name after Garcia stuck a cartoon wolf sticker on it as a joke. When the instrument went back to guitar maker Doug Irwin for repairs, he decided to incorporate the motif in a more permanent way.
Halligan is one of the Deadheads who followed the band for years, travelling from concert to concert.
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The sale of Jerry Garcia's favourite guitar, Wolf, has raised millions of dollars for a civil rights group.