Mitrovic has been struggling with a foot injury in recent weeks and he is currently a doubt for Sunday's Premier League fixture at home against Manchester United.
However, Serbia's all-time leading goalscorer - who has netted 50 times in 76 international appearances - will be heading to Qatar after being named in Dragan Stojkovic's final squad.
Mitrovic scored eight goals in as many games in World Cup Qualifying, including a dramatic 90th-minute winner to secure a 2-1 away victory against Portugal and top spot in their group.
The Fulham striker is expected to lead the line for his nation alongside Juventus star Vlahovic, who will be competing at his first World Cup.
The 22-year-old has scored eight goals in 15 games for a struggling Juve outfit so far this season and he has also been prolific in front of goal for Serbia, netting eight times in 16 international appearances.
Vlahovic is one of 11 players in the 26-man squad who play their club football in Serie A, including another Serbian star in the form of Lazio's attacking midfielder Sergej Milinkovic-Savic.
The 27-year-old, who has been linked with a host of top European clubs in recent months, is an established first-team regular for club and country and is set to be a key asset for Stojkovic in Qatar.
Another important player in Stojkovic's plans in Ajax attacker Dusan Tadic, who has 90 international caps to his name - more than any other player in the squad - and he will captain his nation at this year's tournament.
Elsewhere, Sevilla duo Marko Dmitrovic and Nemanja Gudelj as well as Getafe pair Stefan Mitrovic and Nemanja Maksimovic have all been selected, while Predrag Rajkovic and Srdan Babic - who also ply their trade in La Liga with Mallorca and Almeria respectvley - have also made the cut.
Despite having only played 15 minutes of international football for Serbia, Red Star Belgrade defender Strahinja Erakovic will be heading to Qatar; the 21-year-old is also the only home-based player in Stojkovic's squad.
Former Manchester City defender Matija Nastasic, free agent Uros Spajic and LA Galaxy's Dejan Joveljic - who scored 16 goals in 39 games for the MLS outfit this year - are notable absentees from the 26-man squad.
Serbia will test their mettle against five-time world champions Brazil as well as Switzerland and Cameroon in Group G, and their opening fixture is against the former on November 24.
Serbia's 2022 World Cup squad in full:
Goalkeepers: Marko Dmitrovic (Sevilla), Predrag Rajkovic (Mallorca), Vanja Milinkovic-Savic (Torino)
Defenders: Stefan Mitrovic (Getafe), Nikola Milenkovic (Fiorentina), Strahinja Pavlovic (Red Bull Salzburg), Milos Veljkovic (Werder Bremen), Filip Mladenovic (Legia Warsaw), Strahinja Erakovic (Red Star Belgrade), Srdan Babic (Almeria)
Midfielders: Nemanja Gudelj (Sevilla), Sergej Milinkovic-Savic (Lazio), Sasa Lukic (Torino), Marko Grujic (Porto), Filip Kostic (Juventus), Uros Racic (Braga), Nemanja Maksimovic (Getafe), Ivan Ilic (Hellas Verona), Andrija Zivkovic (PAOK), Darko Lazovic (Hellas Verona)
Attackers: Dusan Tadic (Ajax), Aleksandar Mitrovic (Fulham), Dusan Vlahovic (Juventus), Filip Duricic (Sampdoria), Luka Jovic (Fiorentina, on loan from Real Madrid), Nemanja Radonjic (Torino)