Warren Buffett trails in second place, with his $39bn (£24.5bn) fortune overshadowed by Gates’ enormous $59bn cash pile. The Sage of Omaha was the only member of the top 20 to lose money this year.
Hedge fund manager George Soros, on the other hand, has gained $7.8m to bring his fortune to $22bn and take him into the top ten richest Americans.
Larry Ellison, the chief executive of tech giant Oracle that reported quarterly operating income of $2.7bn on Tuesday, stays in third place with a $33bn fortune.
But further down the list, newer faces are climbing fast. The youngest member of the rich list this year is Dustin Moskovitz, aged 27, who was one of the co-founders of Facebook.
Facebook founder Mark Zuckerberg was the biggest dollar gainer, adding $10.6bn to his wealth in the last year.
The 18 new entrants include Sean Parker, the founder of music service Napster and one of Facebook’s most prominent investors.
Also new to the rich list is John W Henry, the asset manager who owns Liverpool Football Club and the Boston Red Sox, at number 375, and fashion chain Forever 21 founders Jin Sook and Do Won Chang at 88.
Forbes said 70 per cent of the list is made up of entrepreneurs this year – an all-time high. Cost of entry was $1.05bn, up from $1bn last year.
There are 42 women in the Forbes 400 – the same as last year. Wal-Mart scion Christy Walton is the wealthiest, with her family fortune of $24bn making her the sixth-richest American. Six other members of her family are on the list in their own right.
Of the 400 members of the famous list, 262, almost two-thirds, saw their wealth increase over the last twelve months, compared with 217 in the year to August 2010.
Forbes listed Groupon founder Andrew Mason, Twitter’s Jack Dorsey and musician P Diddy as names to watch next year.
The full list is available at www.forbes.com/forbes400.