Carlo Ancelotti's side were held to a 2-2 draw with Valencia on Sunday and remain in third place as a result of that setback.
The UEFA Champions League finalists needed a stunning stoppage-time effort from Cristiano Ronaldo to even salvage a point at the Bernabeu.
That result ensured the Madrid giants are five points behind leaders and city rivals Atletico with three games remaining, although Real do have a game in hand.
Atleti's 2-0 defeat at Levante on Sunday gave their neighbours and Barcelona some slight hope in an enthralling battle for the title.
Barca, three points behind Atletico, also slipped up on Saturday, with strugglers Getafe claiming a 2-2 draw at Camp Nou.
With Atleti - who face Ancelotti's men in the Champions League final on May 24 - and the Catalan giants meeting on the last day of the season, the title is still in their hands.
Wily Italian Ancelotti has not given up hope of more twists, though.
He said: "It's still difficult. It's as I've always said - this league is going to be decided in the last few games.
"There are three games to go that we have to win. It won't be easy, as we play two of them away against teams that have something to fight for.
"We must be clever and prepared. The drive to win is the same as always."
Ronaldo's cheeky backheel was his 50th goal of the season, a sensational landmark that he has achieved for four seasons in a row.
And Valladolid – unbeaten in their last 11 home matches - will have to keep a close eye on the Portugal captain in a game which gives them the opportunity to move out of the relegation zone.
Juan Ignacio Martinez's side breathed new life into the battle to avoid relegation with a precious 1-0 win against Espanyol on Saturday.
Antonio Rukavina picked a great time to score his first goal of the season as he grabbed the winner after 20 minutes at Estadio Municipal Jose Zorrilla.
That was their first win in five games and just a point against Ancelotti's charges would be enough for them to move above Osasuna, Getafe and Almeria into 16th spot with games against already-relegated Real Betis and fellow strugglers Granada to come.
Ancelotti has previously revealed that Spain goalkeeper Iker Casillas will make his first La Liga appearance of the season in place of Diego Lopez to get some playing time ahead of the Champions League final.
But it remains to be seen if Luka Modric (leg injury) is available to return.
Valladolid could be without striker Manucho after the striker sustained a knee injury against Espanyol, while midfielder Fausto Rossi is suspended after picking up his 10th yellow card of the season.
