A referendum on UK membership of the European Union should not be held on the same day as the assembly elections in 2016, the Welsh government has said.
There is speculation the date in May is being considered by the UK government.
A spokesman for Labour's First Minister Carwyn Jones claimed it would limit the ability of pro-EU parties to have a "genuine referendum strategy".
Plaid Cymru peer Lord Elis-Thomas broke ranks with his party on Sunday by saying the move could boost turnout.
His party leader Leanne Wood, along with Mr Jones, has previously said the referendum should not clash with the Welsh assembly and Scottish Parliament elections.
The first minister's spokesman said on Monday: "Our position on this is clear - an in-out EU referendum should be held on a different date from that for the assembly elections.
"The prime minister will be cutting off his nose to spite his face if he pushed for the votes on the same day.
"Labour, the Lib Dems, and the nationalists in Wales and Scotland stand ready to play an energetic part in a 'yes' campaign for a reformed EU.
"However, having the vote on the same day as the Welsh and Scottish elections will limit our ability to have a genuine referendum strategy.
"We know the Tories are divided on the EU, and so won't deliver a credible campaign."
The Conservatives promised in their general election manifesto to hold an in-out referendum on EU membership by 2017, but ministers have played down talk that it could be held before then.
Interviewed two weeks after the election in May, Welsh Secretary Stephen Crabb told BBC Wales he was "relatively relaxed" about the date of the referendum, but was concerned about a "potential clash" with the Welsh assembly and Scottish parliamentary elections.
He told The Wales Report: "I don't really feel it would be healthy for voters going out to vote in those elections with the issue of the EU referendum having dominated the political debate during those election campaigns."