The Welsh Beer & Pub Association said pubs had been left in "limbo", with the last grant covering the period up to the end of March but venues expected to be closed for much longer.
The trade has also called for clarity on when it can expect to reopen.
Chief executive of the British Beer & Pub Association Emma McClarkin said: “Welsh pubs are in limbo, with no clarity on when they can expect to fully reopen nor the financial support they need to continue to survive in lockdown.
“We will continue to work closely with the Welsh Government but our pubs and publicans in Wales are suffering, and cash reserves are rapidly becoming exhausted. If the First Minister wants to prevent pubs from falling at the final hurdle, we must have more support.
"We need a clear indication with dates on when they can expect to fully reopen, as well as a great increase in the level of support they are getting to ensure they can hold on."
In the dark
Geoff Fiddler, operator of one of Wales oldest pubs, the Skirrid in Llanvihangel Cucorney near Abergavenny, called for a detailed timeline for reopening as soon as possible.
He said: "We’ve been kept in the dark for too long with little support. Like many other publicans, we are frustrated by the Welsh Government’s claims that they are providing the most generous support package in the UK when in reality, we are not seeing where this money is going.
"With eligibility restraints around the sector specific Support, so many of us are missing out. Without more information surrounding reopening, or an increase in support, many pubs will certainly struggle to obtain their former glory, place in the community and financial stability.”
First Minister Mark Drakeford has been hesitant to provide a reopening date, suggesting the Government may be able to consider reopening outdoor hospitality at the end of April.
Pubs and restaurants will be given a “reliable date” at a point when “the public health position is safe enough for us to do so,” Drakeford said.
The nation was not “immune” to experiencing the same accelerating case numbers experienced by several European nations that have been plunged into fresh lockdowns, the politician said.
Drakeford announced a further £150m funding would be made available to top-up non domestic rates grants, earlier this month.
Hospitality businesses closed were eligible for a third payment between £4,000 and £5,000 to meet ongoing costs.
The Welsh Government had gone "over and above" the support available from UK Government schemes, making it the "most generous" business support scheme in the UK, Drakeford said.
Boss Brewing co-founder Sara John told The MA without a reopening date for her pub customers, her brewery and bar had “nothing to work towards."
"As an industry, we desperately need some sort of roadmap so that we can plan, and so that we can also see some light at the end of a very dark tunnel," she said.
She said: “England has a roadmap. Scotland has a roadmap. Where’s ours?”