The Azzurri failed to top their FIFA World Cup qualifying group, which leaves them having to beat North Macedonia in the play-off semi-finals on Friday (AEDT) to set up a meeting against Turkey or Portugal five days later.
The draw means one of the two previous European championship winners, Portugal and Italy, will not feature at the 2022 FIFA World Cup in Qatar.
Mancini has named a 33-man squad for the upcoming clash against North Macedonia in Palermo, with Balotelli snubbed for the introduction of Cagliari forward Joao Pedro, who was born in Brazil but has an Italian passport.
Lazio defender Luiz Felipe also qualifies under the same criteria and he features in the Italy squad for the first time, while Federico Chiesa and Leonardo Spinazzola miss out through injury.
Moise Kean, Alessio Romagnoli and Davide Calabria were also notable omissions, with Cristiano Biraghi expected to take Spinazzola's full-back spot and Roma's Nicolo Zaniolo filling in for Chiesa.
Matteo Politano, Stefano Sensi and Pierluigi Gollini, who replaces the injured Napoli goalkeeper Alex Meret, all return, with Manuel Locatelli called up despite testing positive for COVID-19 on Friday (AEDT).
Locatelli's Juventus colleague Leonardo Bonucci has made the squad even though he suffered a reoccurring problem with his calf in Thursday's (AEDT) 3-0 UEFA Champions League aggregate loss to Villarreal.
Alessio Cragno (Cagliari), Gianluigi Donnarumma (Paris Saint-Germain), Pierluigi Gollini (Tottenham), Salvatore Sirigu (Genoa); Francesco Acerbi (Lazio), Alessandro Bastoni (Inter), Cristiano Biraghi (Fiorentina), Leonardo Bonucci (Juventus), Giorgio Chiellini (Juventus), Giovanni Di Lorenzo (Napoli), Emerson Palmieri (Lyon), Alessandro Florenzi (Milan), Luiz Felipe (Lazio), Gianluca Mancini (Roma); Nicolo Barella (Inter), Bryan Cristante (Roma), Jorginho (Chelsea), Manuel Locatelli (Juventus), Lorenzo Pellegrini (Roma), Matteo Pessina (Atalanta), Stefano Sensi (Sampdoria), Sandro Tonali (Milan), Marco Verratti (Paris Saint-Germain); Andrea Belotti (Torino), Domenico Berardi (Sassuolo), Ciro Immobile (Lazio), Lorenzo Insigne (Napoli), Joao Pedro (Cagliari), Matteo Politano (Napoli), Giacomo Raspadori (Sassuolo), Gianluca Scamacca (Sassuolo) Mattia Zaccagni (Lazio), Nicolo Zaniolo (Roma).