It is the first opportunity the public have had to view the Queen's final resting place and see the ledger stone in the George VI memorial chapel which is inscribed with her name.
Anne Daley, 65, from Cardiff, and Grace Gotharg, from Ghana but who lives in London, were the first people in the queue, having arrived at Windsor Castle at 7.30am.
Ms Daley said she felt emotional about entering, adding: "The castle feels like empty, gloomy. Nobody's living in it. You know, you've lost the Queen, you've lost the duke, you lost the corgis.
"It's like when you've sold your house and all the history is gone."
Both Ms Daley and Ms Gotharg had been supporters of the monarchy for a long time.
Ms Daley said: "My father was in the Irish Guards and he did the coronation. So I had an interest from when I was a little girl."
Ms Gotharg added: "I am from the Commonwealth so I knew the Queen before I came here.
"She did a good job as head of the Commonwealth. She was a good woman and a good mother. Everybody loves her."
The pair, who became friends at the unveiling of the Diana, Princess of Wales statue outside Kensington Palace in July 2021, were among the first people to see the Queen lying in state in Westminster Hall.
Ms Daley said: "We waited at Lambeth Bridge on Monday, Tuesday and Wednesday, when we eventually got in at 5pm."
Darren Martin, 43, from near Melbourne in Australia, was the next person in the queue.
He travelled to the UK two weeks ago because of the Queen's death.
Mr Martin said: "I arrived in time to do the queue and camped out for the funeral. I've driven all the way to Balmoral, Holyroodhouse and Sandringham.
"I then had a little bit of a holiday in Sussex and Cornwall. I've come here today for my final send-off before I go back home.
"She was somebody that I admired very much for her service, everything that she did and everything she stood for.
"My grandparents liked the royal family. My grandmother reminded me of the Queen. As a child I fantasised about my own grandmother being the Queen and that stood with my throughout my life."
In 2013 Mr Martin brought his two 85-year-old grandparents to the UK and they visited many of the royal sites.
He said: "It's been quite an emotional experience coming back, but I think it was just something I felt really deeply and strongly about doing.
"It wouldn't have been the same just sitting at home watching everything on TV. I felt strongly enough to be here in person."