Fans yelled "puto" - a homophobic slur - when United States goalkeeper Matt Turner launched the ball up the field and the game was briefly stopped while warnings were given to the supporters.
The "puto" slur means male prostitute but in Mexican Spanish it roughly translates as "faggot," and is widely used to insult someone's masculinity.
The chant has long been a problem for Mexico's Football Federation who have been fined on several occasions and who have urged supporters to stop the practice.
CONCACAF issued a statement after the game at the Allegiant Stadium, which was won 3-0 by the United States saying they "strongly condemned" what it called "discriminatory chanting".
"Chants heard during the game led to the activation of the anti-discrimination protocol by the match officials. Additionally, security staff ejected several fans for engaging in unacceptable behaviour in the stadium," said the confederation, which governs football in North and Central America and the Caribbean.
"These incidents were extremely disappointing and tarnished what should have been a positive occasion to showcase high-quality football in our region," the statement added.
"The Confederation is in the process of urgently establishing further details and reports from security and match officials and will make a further statement in short order".
The multiple fines handed to Mexico over several years, total over $650,000 and they were forced to play without fans against Jamaica in September 2021.
Mexican Football Federation (FMF) introduced a system of online ticket registration and QR codes for attendees at its home games in a bid to identify and ban any offenders and deter use of the chant.
Mexico will co-host the 2026 World Cup along with the United States and Canada and last year, then federation president Yon de Luisa warned they risked losing the hosting rights over the chant.
A heated game ended with both teams having two players each sent off and objects and drinks were also thrown on to the field from the largely Mexico supporting crowd.
Laser pointers were also aimed at Turner and other players on the field.
