Sevilla say they could withdraw from Sunday's Spanish Super Cup if opponents Barcelona name more than three non-European Union players for the match.
The Spanish football federation announced on Saturday there were no restrictions on non-EU players as it classed the game as "not professional".
Only three non-EU players are allowed in each La Liga matchday squad.
It had been thought the Spanish Super Cup was the same but Sevilla said they would object to "an improper line-up".
The annual match pits the La Liga champions against the Copa del Rey winners. Because Barcelona won the domestic double last season, Sevilla qualify as Copa del Rey runners-up.
The match is being played in Tangier, Morocco, and will be contested over one leg for the first time.
A Sevilla club statement read: "Sevilla FC is surprised by the RFEF [Spanish football federation]'s statement 24 hours before the Super Cup.
"It has stated that as many non-EU players as desired can be registered. In their last notice for the 18-19 season, only three were due to be permitted, without exception.
"The club's legal department is studying the matter and should FC Barcelona submit a team with more than three non-EU players, it would present possible ground for withdrawal on account an improper line-up."
Arturo Vidal, Malcom and Arthur are likely to take Barca's three slots. Barca have taken four non-EU players to Morocco, including defender Marlon.
Brazilian Philippe Coutinho is thought to have gained a Portuguese passport in the last few days through his wife, with Luis Suarez and Lionel Messi already having EU citizenship.
Barca boss Ernesto Valverde was not happy with the rule change either. "As for the Supercopa not being a professional competition, I do not know what to tell you," he said.
"I think that Sevilla are professionals and we are professionals. With all the trouble we have had this week with non-EU squad members, they could have warned us before."