Other technology heavyweights in the top 10 include Oracle's Larry Ellison in fourth place with $26bn, Google's Sergey Brin and Larry Page making their first appearance in joint fifth place with $18.5bn, and Dell chief Michael Dell in eighth with $17.2bn.
However, this year's Forbes study showed that having $1bn is no longer enough to make it into the list of the top 400 richest Americans, as 21 Americans shared the 380th spot with a fortune of $1.3bn.
While Gates may still be the richest American, he could soon lose his place as the world's richest man as Mexican telecoms owner Carlos Slim Helú now comes within a mere $500m of his fortune.
Helú's fortune has grown quickly and Forbes admitted that at times he has surpassed the Microsoft chairman.
"Since we released our list of the world's billionaires six months ago, declaring Bill Gates the world's richest man, Helú has closed the gap," said Forbes reporter Matthew Miller. " Who's richer now? It depends on what day it is."
Miller said that, when stock prices were tallied on 31 August, Slim and Gates were within $500m of each other at around $59bn.
"A move of $2 in the share price for Microsoft, or a big cellphone company in which Slim holds a 30 per cent stake, can add or subtract a billion dollars," he said.
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