Six-time champion Serena, 36, beat German world 101 Carina Witthoft 6-2 6-2, while twice champion Venus saw off Italy’s Camila Giorgi 6-4 7-5.
The last time the Americans played each other at a Grand Slam was in the final of the 2017 Australian Open, which Serena won when eight weeks pregnant.
“We’d rather have met later,” 17th seed Serena said.
Serena missed the US Open last year, giving birth to daughter Olympia shortly after the tournament started. She reached the Wimbledon final in July.
Chasing a record-equalling 24th Grand Slam singles title, Serena unleashed 30 winners and 13 aces on her way to victory over Witthoft and sealed victory when the German netted a forehand.
While Serena wrapped up her win in one hour seven minutes, it took Venus an extra 45 minutes to beat Giorgi.
“Obviously, it’s early in the tournament, so both of us are going to be looking forward to continuing to play better,” 16th seed Venus said.
During the most recent Williams sisters meeting at Indian Wells in March Venus won during Serena’s first tournament back, but the 36-year-old showed how much sharper she has got since then.
The Wimbledon finalist said: “It’s obviously a tough match. It’s so young in the tournament. It’s not the end of the world. We would have rather met later but we’re both going to come out per usual and do our best.
“We make each other better. We bring out the best when we play each other. I think we’re used to it now.
“I never root against her, no matter what. So I think that’s the toughest part for me. When you always want someone to win, to have to beat them. I know it’s the same thing for her.”
Another blockbuster clash will see defending champion Sloane Stephens take on former world number one Victoria Azarenka, who has found her form in New York.