Microsoft founder Gates retained the top spot for the 20th year in a row - now worth a net $72 billion, up from $66 billion a year ago.
The 57-year old, who has given away about $28 billion over the years, has overtaken Mexico's Carlos Slim to become the world's richest person again.
Investor Warren Buffett is the second richest American, with a net worth of $58.5 billion, having grown his fortune by $12.5 billion since last year.
Buffett's Berkshire Hathaway firm bought ketchup-maker H.J. Heinz for $23.2 billion in June and Berkshire's shares have risen 34 per cent.
Larry Ellison, head of American multinational Oracle, took the number three spot with $41 billion.
Four members of the Walton family, who have made their money through department store chain Wal-Mart, featured in the top 10.
Mark Zuckerberg returned to the Top 20 after missing out last year. Facebook shares have soared recently, boosted by strong mobile ad growth, and the 29-year old has in turn grown his wealth from $9.4 billion to $19 billion.
Only three women made it into the top 20 - Wal-Mart's Christy and Alice Walton, and candy company owner Jacqueline Mars.
Forbes said the minimum net worth needed to make the 400 list this year was $1.3 billion.
"The last time it was that high was in 2007 and 2008, before property and stock market values began sliding."
More than 60 American billionaires failed to make the cut.
Top 10 richest Americans
1. Bill Gates $72 B
2. Warren Buffett $58.5 B
3. Larry Ellison $41 B
4. Charles Koch $36 B
5. David Koch $36 B
6 Christy Walton & family $35.4 B
7. Jim Walton $33.8 B
8. Alice Walton $33.5 B
9 . S. Robson Walton $33.3 B
10. Michael Bloomberg $31 B