Following tests on Monday, Madrid have announced Kroos has an issue in the gluteal region, though did not reveal how long the 30-year-old will be sidelined.
“Following tests carried out today on our player, Toni Kroos, by the Real Madrid medical services, he has been diagnosed with a muscular injury to the left gluteus medius," a statement from the club read.
"His recovery will continue to be assessed."
Zinedine Zidane only has three days to prepare his squad for Valladolid's arrival at Santiago Bernabeu, with a tough away trip to Levante set to follow that clash on October 4.
Kroos may also be unavailable for a trio of upcoming international fixtures. Germany play Turkey in a friendly on October 7, followed by Nations League games against Ukraine and Switzerland during the break.
Madrid will return to action later in the month with a home fixture against Cadiz, before preparations begin for the first Clasico of the season against arch-rivals Barcelona on October 25.
The Blancos can ill-afford to lose a talismanic performer like Kroos for an extended period of time, especially given the fact that they have yet to strengthen their squad in the summer transfer window.
The likes of Brahim Diaz, James Rodriguez, Gareth Bale and Sergio Reguilon have all left the Bernabeu, but Madrid have decided against investing in new talent amid the financial uncertainty caused by the coronavirus pandemic.
Zidane was quizzed on Madrid's lack of activity in the market after watching his side pick up their first three points of the new La Liga season at the weekend, to which he responded: "No. We spoke about this before. I'm happy with the squad that I've got.
"We are what we are and we're going to try to have a successful season."
Club captain Ramos added on the need for Madrid's current first-team stars to continue giving their all to the collective cause regardless of any incomings and outgoings: "We have to show our faces with those of us who are already here, but our lives don't depend on one signing or another."