Smit, who returned to the starting line up in place of Bismarck du Plessis, came off the bench in the Springboks' final pool game against Samoa but barely spent any time on the field after being sin-binned for a deliberate knock down within minutes of running on.
Botha, who had said he was "100 percent fit" to face the Tri-Nations champions, injured himself in training on Wednesday and was not considered for the side named by coach Peter de Villiers on Thursday.
"That was the worst part of my day yesterday. At training when Bakkies came down in the first lineout he injured himself again," De Villiers told reporters.
"Sadly for myself, and I think for Bakkies too, it might mean that his tournament will be over now.
"To look in his face last night wasn't the best thing to do, but that's reality, that's life, this is what we get and we just have to move on."
De Villiers said the 32-year-old had aggravated his chronic Achilles injury, which had forced him to play just a bit part in the Springboks' two pool matches against Fiji and Namibia.
Botha had also suffered a slight hamstring strain, but de Villiers said he had recovered from that injury.
"It was actually fine for him to go. It's just one of those accidents that you get - not very good support when he came down; he landed with several tonnes of weight on that foot (and) when he came down he landed badly, so the Achilles flared up again."
De Villiers said they probably would not seek a replacement for Botha, should he be definitively ruled out, until after the quarter-final.
"We'll play it by ear and we'll see what happens this weekend and then we will make a decision on Monday."
The impressive Danie Rossouw retained his place in the second row alongside vice-captain Victor Matfield
Botha was not the only injury concern for de Villiers as he named a vastly experienced side, which will again be led by hooker John Smit, with winger JP Pietersen likely to have undergo a fitness test on a knee injury.
Both Pietersen and fellow winger Bryan Habana had been injury concerns heading into the match.
"JP is not one hundred percent as yet, but we'll give him all the time that we can to prove his fitness and we just hope that he will get through," de Villiers added.
"We've got a little tiger in (Francois) Hougaard so he will be the one who will step in there (if Pietersen is unfit)."
De Villiers' team is the most experienced Springboks side to take the field, with a total of 836 caps. The side that played Wales in their World Cup opener had 815 caps.
The starting side on Sunday includes eight players who began the 2007 World Cup final victory over England and three others who were part of the Springboks' winning squad.
Only fullback Pat Lambie, flyhalf Morne Steyn, flanker Heinrich Brussow and number eight Pierre Spies were not involved in the 2007 campaign, though Spies was selected in the squad but had to withdraw with illness.
"Certainly it's quite an experienced team, but it only really counts if we can get the business done on Sunday," said Smit.
Inside centre Jean de Villiers, who came on for Frans Steyn against Samoa, replaced Steyn for Sunday's match after the utility back was ruled out of the tournament with a shoulder injury.
"I could understand his excitement last week when he came off the field against Samoa and he promptly told me it was the first World Cup game he's been able to finish," Smit said of de Villiers selection.
"He's pretty excited and I could see it in his enthusiasm this week."
Team: 15-Pat Lambie, 14-JP Pietersen, 13-Jaque Fourie, 12-Jean de Villiers, 11-Bryan Habana, 10-Morne Steyn, 9-Fourie du Preez, 8-Pierre Spies, 7-Schalk Burger, 6-Heinrich Brussow, 5-Victor Matfield, 4-Danie Rossouw, 3-Jannie du Plessis, 2-John Smit (captain), 1-Gurthro Steenkamp
Replacements: 16-Bismarck du Plessis, 17-CJ van der Linde, 18-Willem Alberts, 19-Francois Louw, 20-Francois Hougaard, 21-Butch James, 22-Gio Aplon