That's in response to what they described as the country's "deepening military relationship" with Russia.
South Africa hosted joint naval exercises with Russia and China earlier this year.
It also plans to host a summit of leaders from the BRICS group of nations.
Russian President Vladimir Putin is invited, despite being subject to an arrest warrant issued by the International Criminal Court.
In the letter to U.S. Secretary of State Antony Blinken and other senior officials, the lawmakers suggested South Africa is in danger of losing its benefits under the African Growth and Opportunity Act, or AGOA.
That's Washington's flagship trade program.
It grants qualifying countries' exports preferential access to the U.S.
Johannesburg is due to host the AGOA Forum later this year .
African nations will be seeking to extend the program, scheduled to expire in 2025.
South Africa's exports to the U.S. under AGOA reached nearly $1 billion in the first three months of this year, making it the second biggest beneficiary after Nigeria.
In the U.S. letter, dated June 9, lawmakers said they were "seriously concerned" that hosting the summit in South Africa would "serve as an implicit endorsement of South Africa's damaging support for Russia's invasion of Ukraine."
South Africa has declared its neutrality in the Ukraine conflict - which Russia calls a special military operation.
However the letter also appeared to back up an accusation by the U.S. ambassador to South Africa that a sanctioned Russian vessel collected weapons at a South African naval base last year.
South African officials say they are not aware of such an arms transfer and have launched an independent inquiry.
Referring to the letter, South African foreign ministry spokesman Clayson Monyela said on Twitter: "There is no decision by the State Department/White House to move the AGOA Forum from SA."
South Africa's Department of Trade and Industry said it was not planning to respond publicly to the letter.
