Alcacer played the full 90 minutes for Spain in their 3-0 Euro 2016 qualifying win over Belarus last Saturday but did not feature in Tuesday's 1-0 friendly defeat to Germany.
The 21-year-old has scored four goals for Valencia this season but is now likely to miss out on Sunday's derby with city rivals Levante after his club confirmed he had sustained a grade one injury to his right hamstring.
Alcacer's prospective absence could see Alvaro Negredo - who has made only three substitute appearances in the league since joining Valencia on loan from Manchester City - lead the attack this weekend.
Third-placed Valencia are seeking to keep in touch with leaders Real Madrid following an encouraging start to the season.
They will also be aiming to atone for a 2-0 loss at Levante in last season's corresponding fixture.
