William Graham and Mohammad Asghar will have to enter an open list to compete against other hopefuls.
Mr Graham, an AM since 1999, told BBC Wales: "The process is ongoing.  It's all under discussion."
Mr Asghar said he was "not disappointed", but declined to comment further.
Mr Graham is the Welsh Tories' Shadow Economy Minister and a former whip who chairs the enterprise and business committee.
Mr Asghar was elected as a Plaid Cymru AM in 2007, and defected to the Tories in 2009.
The decision was taken at a regional board meeting on Wednesday night.
Twenty of the 60 assembly members are elected on the basis of regional lists which offer a form of proportional representation, with four seats available in each of five regions.
This is a blow to William Graham and Mohammad Asghar, who now find themselves in a battle to become the top candidates on the regional list in south east Wales.
The local board clearly wants to give others the chance, and the two AMs will be in the mix with a range of other candidates at a hustings attended by the wider local membership, and not just the board.
Whatever the overall result next May, there will be major changes in personnel at the Senedd, particularly in Labour ranks with so many of its AMs not standing.
It will be interesting to see if there will be significant changes among Tory ranks as well.
Two Conservative AMs have failed to win automatic re-selection as regional candidates for south east Wales in the 2016 assembly election.