The 25-year-old hurt his right foot in a friendly 4-3 win over second-division Fortuna Duesseldorf on Tuesday and Dortmund's team doctor Markus Braun has said he is expected to be out for two weeks.
The centre-back only made his return in Friday's 2-1 win at Eintracht Braunschweig after two-months out after injuring his right heel against England in an international friendly.
A scan on the ankle showed the November injury has fully healed, but the new ligament strain will need a fortnight's rest.
With Dortmund third in the table, Hummels will miss Saturday's league match at home to Werder Bremen, plus two matches against Eintracht Frankfurt within four days, first in Tuesday's DFB-Pokal quarter-finals, then in the Bundesliga.
Hummels' injury is more bad news for Dortmund coach Jurgen Klopp with both centre-back Neven Subotic and winger Jakub Blaszczykowski out for the rest of the season with torn knee ligament injuries.
The loss of Hummels means Klopp will again be down to his fourth-choice centre-back in either veteran Manuel Friedrich or teenager Marian Sarr alongside Greece centre-back Sokratis.
There was some good news for Klopp on Tuesday as attacking midfielder Jonas Hofmann scored twice and new-signing Milos Jojic made his debut in the win over Fortuna.