The English Football League says a quarter of players at its member clubs "do not intend to get a vaccine" against Covid-19.
Tighter protocols at first-team training grounds and more regular testing have been enforced.
It follows the postponement of more fixtures scheduled to be played on Saturday across all three divisions.
Updated guidance on the minimum number of available players required to fulfil a fixture has also been passed on.
Clubs will be expected to play a fixture where they have 14 players, including a goalkeeper, available from the their registered squad list.
Vaccine data collected by the EFL in November showed 75% of players were either fully vaccinated, had had a single jab or intended to be vaccinated.