The Germany forward was taken off in the second minute of stoppage-time in his country's 2-1 victory in Sunday's UEFA Euro 2016 qualifier with Scotland after appearing to injure himself when attempting to pull away from a Charlie Mulgrew challenge.
And Dortmund have now confirmed that Reus has sustained a partial tear of ligaments in his left ankle.
The injury is strikingly similar to the one he suffered while on Germany duty in a friendly win over Armenia in June, a blow that kept him out of a successful World Cup campaign for Joachim Low's men.
Reus, 25, will now be on the sidelines for the next four weeks as Dortmund aim to lay down an early marker in the race for the Bundesliga title.
Dortmund posted a picture of Reus on their Twitter account with the caption: "Lateral ligament partial tear and strain of the transverse ligament in the left ankle. Approximately 4 weeks rehab. Get well soon."
The former Borussia Monchengladbach man is likely to miss five Bundesliga matches, including the season's first Revierderby with Schalke, and UEFA Champions League clashes with Arsenal and Anderlecht.
