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With a game in hand, Hansi Flick's Barca are top on 66 points, three ahead Real in second and nine clear of third-placed Atletico. If Barca win against Betis later on Saturday, they will extend their lead to six points with eight games to go.
Real Madrid dominated possession throughout the game but wasted too many chances, with Valencia goalkeeper Giorgi Mamardashvili putting in a brilliant performance between the posts, including blocking Vinicius' first-half penalty.
After Kylian Mbappe was fouled inside the box in the 12th minute, Brazilian forward Vinicius, who had missed a penalty in Real's Champions League last-16 clash against Atletico three weeks ago, struck a feeble effort low to the goalkeeper’s left that Mamardashvili pounced on.
In the following play, defender Mouctar Diakhaby opened the scoring for Valencia with a header from an Andre Almeida corner in the 15th minute but almost gifted Real the equaliser when he put the ball into his own net six minutes later.
However, he was rescued by VAR who spotted an offside in the build-up that ruled it out and Mamardashvili made a string of saves to keep Valencia's lead before the break, with Mbappe denied several attempts.
Vinicius scored the equaliser from a corner in the 50th minute and pushed for the winner in the second half, with the Georgian keeper, on loan at Valencia from Liverpool, continuing to make stunning saves to frustrate Real Madrid.
Valencia held on and took advantage of a quick counter five minutes deep into stoppage time as Hugo Duro's header secured their first win away from home in LaLiga this season.