Barnes and Noble said the previous record was held by Dan Brown for his 2009 bestseller "The Lost Symbol," which is to be made into a film expected to star Tom Hanks.
"Watchman" surpassed E L James' mommy-porn erotica "Grey" as the number one bestseller and is on track to be the company's bestselling book of 2015, the company said.
Its global release on Tuesday was one of the biggest events in modern publishing history, more than half a century after her classic "To Kill a Mockingbird" made its debut.
Sales for Lee's first novel had doubled since then and "Mockingbird" is now the number two bestselling book, Barnes and Noble added.
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It is considered a 20th century classic that defined racial injustice in the Depression-era South of the United States and became standard reading in classrooms across the world.
The literary world was upended when HarperCollins announced it was publishing a second novel, seemingly discovered from Lee's safe-deposit box in still-unclear circumstances.
Lee wrote the manuscript in the late 1950s, but her then-editor suggested she recast the book from the childhood perspective of Scout, which in turn became "Mockingbird."
Barnes and Noble is a Fortune 500 company, with 648 stores in 50 states and it also sells online.