Venus claimed the upper hand in the 2001 final - the last time she won the tournament - and the 2005 round of 16.
The Williams sisters have not met at such an early stage of a Grand Slam since their first professional meeting at the 2nd round of the 1998 Australian Open. It will be their sixth face-off in NY.
Venus emerged a 6-2, 6-4 victor, something her sister admitted she barely remembers, but Serena also revealed she reckons their individual success helps them both reach higher levels.
French player Alize Cornet said the decision to ban Serena Williams from wearing a catsuit at future French Opens was "10,000 times worse" than her on-court reprimand at the US Open. "I'm pretty pumped to get through", added Venus, playing in her 80th Grand Slam event.
"At least this time, " Venus told the crowd, "it'll be fair".
'The best part is we bring out the best in each other.
Manchester United unveil pink away kit for 2018/19 campaign
We do a lot of possession and try to press high. "Some footballers hold the ball too much, Hazard most of all". New boss Sarri has previously said it takes defenders longer to adapt to his unique style of play.
Serena, meanwhile, put together a 30-10 edge in winners, then declared her serve much better than it's been of late. So I think that's the toughest part for me: "When you always want someone to win, to have to beat them", Serena said. "Whatever, it will work".
She said: 'I think just the umpire was probably overwhelmed by the situation.
Defending champion Sloane Stephens avoided an early exit, recovering from a set down to defeat Ukrainian qualifier Anhelina Kalinina 4-6 7-5 6-2.
Stephens triumphed after 2 hours and 46 minutes on Arthur Ashe Stadium and will next face two-time Australian Open champion and former world number one Victoria Azarenka.
"Bernard Giudicelli lives in another time", she said. The younger sister, on the hunt for her record-tying 24th Grand Slam singles title, delivered a strong serve and finished off the young German behind 13 aces and 30 winners. "Every year it's such a tough field in Wuhan so I'll be ready to compete hard amongst some really great players".
Tournament officials said they regret that a player was given a code violation for changing her shirt on the court during a match, clarifying its rules on when that is allowed.