Football Australia (FA) confirmed on Thursday that the long-awaited tournament will feature 16 clubs from each state as well as the ACT.
The NST will kick off in October 2025.
In a structure akin to the Champions League format, teams will be split across four groups of four and play home-and-away group fixtures.
The top two sides will advance to a knockout stage that culminates in a final in early December.
Eight clubs from NSW and Victoria - APIA Leichhardt, Avondale, Marconi, Preston Lions, South Melbourne, Sydney Olympic, Sydney United 58 and Wollongong Wolves - will be recognised as foundation members.
Only Avondale were not previously a member of the defunct National Soccer League (NSL).
Sydney United 58 players thank supporters after their 2022 Australia Cup final loss to Macarthur FC. (Dan Himbrechts/AAP PHOTOS)
These eight foundation clubs will be supplemented by eight premiers of the existing National Premier League competitions in NSW, Northern NSW, Victoria, SA, WA, the ACT, Tasmania and Queensland.
"The introduction of the national second tier is a landmark moment for football in Australia," Football Australia chief executive James Johnson said in a statement.
"Following productive discussions with our eight foundation clubs and consultation with other stakeholders, we are committed to creating a competition that not only elevates our game but also enriches the football community at all levels.
"The national second tier will provide a new platform for iconic clubs to showcase their talent and connect with fans nationwide."
The competition's name and trophy will be unveiled in the run-up to the start date.
FA maintains the long-term aim is for the second tier to move away from the Champions League style structure and into a full-blown home-and-away season.
It remains unclear how, when, or if promotion to the A-League Men may be introduced.
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