Nine-time tournament finalist Chile asked FIFA to investigate allegations that Ecuador used ineligible right-back Byron Castillo in its successful qualifying campaign.
The Federacion de Futbol de Chile (ANPF) said it had proof that Castillo was born in Colombia and not Ecuador, as stated on his official documents.
Castillo appeared eight times in qualifying, including in a goalless draw with Chile in September and a 2-0 victory in November.
La Roja finished seventh, meaning they just missed out on a play-off spot, but felt Ecuador should be expelled from the World Cup at their expense.
However, after looking into the claims made by ANPF, world football's governing body FIFA rejected the appeal last month and confirmed Ecuador will keep its place in Qatar.
Chile is continuing to push its case, though, with its general secretary Jorge Yunge confirming on Friday (AEST) that a fresh appeal will be lodged.
"We are completely sure about the conclusions of our investigations," Yunge said in a statement.
"It's absolutely clear the player uses an adulterated Ecuadorean document. What is at stake here is not just a place in the 2022 World Cup, but the entire principle of fair play."
Ecuador has been drawn in Group A at the World Cup, which begins in November, alongside Senegal, the Netherlands and host Qatar.