The Ospreys wing was set to make his Wales bow this weekend after being named in Warren Gatland's side on Tuesday.
But sadly for the 21-year-old, who missed last year's Six Nations and the rest of the season after suffering a bulged disk in his back, injury has struck again. Liam Williams comes into the side with Ashley Beck moving up to the bench.
"Eli pulled up this morning with a hamstring strain. We're not sure whether it's a grade one or grade two," said Howley.
"He's going to have further scans and we'll await the results. It's a blow to us. He's in good form and I feel so desperate for the player himself. He's trained particularly well but it gives another player an opportunity."
Howley added: "Eli is pretty disappointed. It happened last year and you feel for players but he's a fantastic kid and he's added so much to our environment. We have to focus on the weekend now."
Walker's injury aside, Wales have a fully fit squad for Saturday's clash against a Springbok starting XV which boasts 667 Test caps. Howley admitted Heyneke Meyer is able to call on a great deal of talent but says Wales are more than up for the challenge.
"We're really enthusiastic, as you should for the first game of the autumn against one of the best sides in world rugby," said the former scrum half.
"When you see the side that was selected yesterday, it's pretty impressive. They have world class players but we feel in our 11 British Lions, we have experience. I'm sure there will be a large amount of kicking - we'll have to dominate the aerial battle but it's going to be a great game."
Meyer's key selection calls saw him pluck for Jaque Fourie in the centre alongside captain Jean de Villiers with Pat Lambie at full back. Eben Etzebeth partners Flip van der Merwe at lock meaning there is no place in the match day 23 for Bakkies Botha.
"I thought they'd bring back Jaque Fourie, his partnership with de Villiers is going to be crucial to the way they play," said Howley.
"Whether it's Lambie or Kirchner at full back, I've no doubts there will be an exchange in terms of kicking early on. It's important we're accurate and when we kick the ball, there's a 50/50 challenge.
"You have to earn the right to play against these sides. It's going to be an arm wrestle and that's the nature of international rugby when two world class sides come together."