It is the third time he has won Paralympic handcycling gold.
Zanardi passed Australian Stuart Tripp in the H5 time trial at Pontal to win the gold medal by 2.74 secs with a time of 28m36.81s.
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Zanardi - who raced in 41 F1 races in the 1990s - is competing in two events in Rio. He is back in action on Friday as part of a three-man Italian team in the H2-H5 mixed relay.
Zanardi won two golds at London four years ago after taking up the sport in 2007. He also has six World Championship gold medals.
Thursday marks the 15th anniversary of the tragic accident at EuroSpeedway Lausitz that saw both his legs amputated.
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