Media have reported that up to a dozen players have declined the shots, including Gold Coast Titans' Bryce Cartwright and three Canberra Raiders.
Prime Minister Scott Morrison waded into the issue on Wednesday, saying that players should not be able to train or play without the vaccinations.
"When I was social services minister I started the 'no jab, no play' rule into the childcare facilities," Morrison told local radio station 2GB.
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"And I think the same rule applies there — no jab, no play."
An NRL spokesperson said players who refused the shot would be dealt with on a case-by-case basis in consultation with their club and NRL medical staff.
A spokesman for the Rugby League Players' Association said the union encouraged players to get the shots but added that they had the option to sign a waiver which would allow them to train and be available for selection while their case was dealt with by the NRL.
The RLPA said the union and the NRL would clarify the vaccination policy after consulting with stakeholders.
No vaccine has been developed for COVID-19, though drugmakers are rushing to develop treatments and vaccines for the disease which has killed over 260,000 people worldwide, infected more than 3.8 million and ravaged economies globally.