Portland Thorns and North Carolina Courage players took a knee during the national anthem as the National Women’s Soccer League began on Saturday.
The players also wore T-shirts reading “Black Lives Matter” before kick-off.
The teams said it was to protest against “racial injustice, police brutality and systemic racism against black people and people of colour”.
Ex-NFL player Colin Kaepernick began kneeling during the US anthem in 2016 in protest against racial injustice.
The joint statement added: “We love our country and we have taken this opportunity to hold it to a higher standard.
“It is our duty to demand that the liberties and freedoms this nation was founded upon are extended to everyone.”
US star Megan Rapinoe, who has opted out of the tournament, said on social media: “You love to see it.
“You love to see these women using their voice, demanding better for America, and for black people and people of colour.”
The protest has taken renewed significance since the death of George Floyd in the United States.
Floyd, a 46-year-old unarmed black man, died after being restrained by a white Minneapolis police officer on 25 May.
Saturday’s match in Utah was the first time a major North American league has returned to action since the start of the coronavirus pandemic, which halted sports in March.
Courage won the game 2-1.
Later on Saturday, players from Washington Spirit and Chicago Red Stars also joined the protest.
In an emotional moment, Chicago defender Casey Short was embraced by captain Julie Ertz as they knelt during the anthem.