The Congressional Friends of Ireland, a group in the US Congress which supports and promotes peace and reconciliation in Northern Ireland, has written to the prime minister warning that it will oppose any US-UK trade deal if it risks undermining the Good Friday Agreement.
Any trade deal that put that in jeopardy would be "highly unlikely", it said.
The American politicians told Mr Johnson: "The United States, and the more than 33 million Americans with Irish ancestry, share a genuine interest in the continued success of the Good Friday Agreement and the hard-won peace and prosperity it has brought to so many.
"As you know, America is guarantor of that international peace accord. That is why we strongly oppose any unravelling of the historic treaty or a return of a physical border on the island of Ireland under any circumstances."
The added: "We share the view expressed by other leaders in Congress that any weakening of the Good Friday Agreement or threat to the 310 mile seamless border would make the prospect of a future US-UK trade deal highly unlikely."
The letter also raises concerns about the "tension-filled summer" in Northern Ireland, saying the peace process is "still fragile and needs to be nurtured".
It added: "We will oppose any US-UK trade deal if the Good Friday Agreement is undermined."
The border that separates Northern Ireland and the Republic has been a key part of the Brexit negotiations.
The UK and European Union agreed that whatever happens as a result of Brexit there should be no new physical checks or infrastructure at the frontier.
If Britain does leave the 27 nation bloc, the 310-mile Irish border will represent the only land border between the UK and the EU and is likely to end up with different rules and standards to its neighbour.
The backstop is a position of last resort, to maintain a seamless border, but it would see the UK retaining a very close relationship with the EU for an indefinite period.
Mr Johnson has called for its abolition, saying the current solution would mean "signing away" the UK's "economic independence".