Written less than two years before her death, it shows the late Princess of Wales – who died aged 36 in a Paris car crash in 1997 – expressing her desire to teach her sons "the importance of communication on a deeper level".
The handwritten correspondence, dated November 1995, was sent by Diana to royal supporter Michael Barratt after he wrote to her following her landmark BBC Panorama interview.
The two-page letter is now being offered for sale by Reeman Dansie's Royalty, Antiques and Fine Art auction, accompanied by its original envelope bearing Diana's handwritten name and address.
The letter sheds new light on Diana's reflections following one of the most consequential periods of her life, when she publicly addressed the collapse of her marriage to the then-prince now King Charles, 77.
According to the auction house's description, Diana used the letter to express gratitude for the support she received following the interview and reflected on the future she hoped to build for her sons.
The auction catalogue states Diana wrote about looking forward to the future and "sharing with and teaching William and Harry the importance of communication on a deeper level".
The letter was written shortly after Diana's widely watched and now infamous Panorama interview with Martin Bashir, in which she spoke openly about the breakdown of her marriage.
During the chat interview, she confirmed infidelity had contributed to the collapse of her relationship with Charles, in what has become one of the defining royal television moments of the 20th century.
Diana concluded her letter by thanking Michael for writing to her.
She wrote: "With my best wishes, Yours sincerely, Diana."
The correspondence is expected to sell for between £3000 ($A5600) and £5000 ($A9,400).
Its contents have attracted renewed interest because of the ongoing estrangement between William, 43, and Harry, 41. The brothers, once regarded as a close partnership within the Royal Family, have experienced a well-documented breakdown in relations over recent years.
Tensions intensified after Harry and his wife Meghan, Duchess of Sussex, 44, stepped back from royal duties in 2020 and relocated to North America to raise their two children.
In his memoir Spare, Harry alleged William physically confronted him during an argument concerning Meghan.
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