Villa was a surprise inclusion in Julen Lopetegui's latest squad after three years away, and he made a brief substitute appearance against Italy on Saturday.
Villa came on in the closing minutes to win his 98th cap in the 3-0 victory in Madrid, replacing the impressive Isco at the Santiago Bernabeu.
But Spain confirmed he will not travel for the match against against Liechtenstein on Tuesday.
"David Villa has suffered a slight elongation in his adductor muscle in training," a Royal Spanish Football Federation (RFEF) statement read.
"At the end of the session, Villa went with the medical team of the RFEF to the university hospital Sanitas La Moraleja, where he was given an MRI."
Villa has scored 19 goals and added nine assists for NYCFC duing the 2017 MLS campaign, which has sees his side sit second in the league's Eastern Conference.