Goals from Filip Djordjevic, Aleksandar Kolarov and Lazar Markovic at the Cardiff City Stadium gave Serbia the win as they completed the double over their Welsh counterparts in Group A of UEFA's FIFA World Cup section.
The home side rarely threatened the Serbia goal and a poor performance from Chris Coleman’s men saw them lose their sixth game of the campaign on Tuesday night.
Wales boss Coleman recalled Danny Gabbidon, Andy King, Andrew Crofts, and Sam Vokes to his starting XI but Bale was on the bench after coming through training on Monday in good condition.
Serbia fielded a strong line-up with Milan Bisevac, Ivan Radovanovic, Dusan Tadic and Filip Djuricic all coming into the starting line-up after Friday’s 1-1 draw with Croatia.
And after a quick start, the visitors took the lead inside eight minutes when Andrew Crofts kept Djordjevic onside and he headed home under no pressure inside the six-yard box.
Serbia continued to look dangerous from set-pieces with both Dusan Tadic and Branislav Ivanovic going close with efforts from free-kicks.
Wales did settle, with Aaron Ramsey creating a number of half-chances but a wonderful strike from Kolarov in the 38th minute doubled Serbia’s advantage when he let fly from distance to give Myhill no chance in the Wales goal.
The game was wrapped up in the 55th minute when Markovic reacted quickest following two efforts from Tadic and he side-footed the ball into the net.
Wales continued to be overrun but the home crowd did have something to cheer about when Bale was introduced into the game just before the hour mark.
The Real Madrid star went close on two occasions with testing efforts as he showed his superior skills despite a long lay-off this summer.
Bale again tested Vladimir Stojkovic in the Serbia goal with a long-range free-kick, but the Partizan Belgrade man reacted well to tip the ball over.
But Coleman's side came out empty-handed and slumped to the bottom of the group, courtesy of Scotland's win over Macedonia.
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