Aaron Ramsey had given Chris Coleman's Dragons a first-half lead from the penalty spot in Belgrade.But Mitrovic - who had been starved of service for much of the game - struck with 17 minutes left.
Aleksander Prijovic backheeled the ball into the Newcastle striker's path, and Mitrovic touched it past a defender before firing past Wayne Hennessey.
He had also scored against Wales in Cardiff in November, and has now scored five goals during this World Cup qualifying campaign.
Serbia remain on course to reach the finals - they're joint top of Group D - but Wales now only have a slim chance of qualifying.
Republic of Ireland's own hopes of making it to Russia next summer were boosted as they came from behind to draw with Austria in Dublin.
Daryl Murphy came from the bench in the 56th minute with his side trailing, and Ireland's improved showing produced an equaliser for Jon Walters five minutes from time.
Meanwhile, the USA drew 1-1 with Mexico at Estadio Azteca. DeAndre Yedlin played the full game.