Strikers Siyabonga Mkhwanazi, Dumisani Ngwenya and midfielders Effort Sithole and Bafo Biyela were all left behind in Durban because they have suffered injuries.
Mkhwanazi and Ngwenya, who have netted 17 goals between them this season, and Sithole, missed the Nedbank Cup semi against Mamelodi Sundowns.
Biyela was substituted in the second half against Sundowns and his place was taken by former Kaizer Chiefs midfielder Nhlakanipho Mkhize.
Usuthu manager James Dlamini confirmed the developments, but said he was hopeful they were going to come back with full points.
Dlamini added that they have raised their concerns with the PSL regarding them being fixtured in three away matches in succession. They played Free State Stars, BidVest Wits and now this Ajax tie.
“This is taking its toll on the players and we don’t know how this can be averted in the future,” he said.