The Shepherd’s Crown, the 41st novel in his Discworld series, went on sale in the UK and Commonwealth at midnight BST (23:00 GMT Wednesday).
In the UK, fans gathered for midnight openings at stores in London, Oxford and Newcastle. More shops will open earlier than usual on Thursday.
The Shepherd’s Crown will be published in the US on 1 September.
The release will be an emotional moment for many fans. One book shop in Perth, Australia, is giving away a free packet of tissues to every customer “because this really is goodbye”.
Sir Terry died at the age of 66 in March, eight years after being diagnosed with Alzheimer’s disease.
He built up a huge following for the Discworld series, which started as an affectionate parody of fantasy novels and became a vast satire on modern life.
Shops opening at midnight included Waterstones in Piccadilly, central London, where the author’s friend Rob Wilkins read an extract to 200 ticket-holders.
Mr Wilkins earlier told BBC News: “It was a hard book to complete because Terry’s health was declining in the last year. But he was still enjoying the writing.”
The Shepherd’s Crown is the fifth Discworld novel to feature young witch Tiffany Aching.
As well as the midnight openings, some shops elsewhere in the UK had breakfast openings and others will host special readings during the day.