Higuain was omitted for the clashes against Venezuela and Uruguay last month - both ended in draws - and he has once again been left at home for the remaining two matches.
The 29-year-old has been in decent form this season, scoring two goals from three Serie A appearances, but he has failed to impresses national manager Jorge Sampaoli and will have to watch qualifying unfold from home.
Paris Saint-Germain forward Javier Pastore has also been left out of the squad after picking up a back injury whilst West Ham United's Manuel Lanzini is absent with a knee problem.
Sevilla's Guido Pizarro and Joaquin Correa as well as Atletico Madrid's Augusto Fernandez have been dropped although there is a place for the uncapped German Pezzella of Fiorentina.
Despite leaving out Higuain and Pastore, Argentina still have a wealth of attacking talent in their squad in the shape of Lionel Messi, Paulo Dybala, Mauro Icardi and Sergio Aguero.
La Albiceleste are currently fifth in the group, just outside of the four automatic qualifying places, and they face fourth-placed Peru on October 5 before going up against Ecuador, who are in eighth, on October 10.