Counting officers were told to ignore the votes of people who wrote "No" next to the box which said: "I agree that there should be a Welsh assembly," it emerged last night.
But a senior electoral officer said she was told to count the votes of people who put "Yes" next to a second box on the ballot paper which said: "I do not agree that there should be a Welsh assembly."
Alison Childs, the principal electoral officer for Rhondda Cynon Taff council, said she believed her counterparts around Wales had rung the office of Professor Eric Sunderland, the Chief Counting Officer, for clarification and had been given the same guidance.
Written rules sent out in advance gave different advice. They said if voters' intentions were clear their papers should be counted.
Ms Childs said electoral officers had complained to the Welsh Office in advance of the vote that the two-question ballot paper would cause confusion. In the event there were 4,000 spoiled papers - more than half the narrow 6,700 majority in favour of an assembly.
"We said there was obviously a huge problem. We told them that in advance because of the wording of the ballot paper. I believe every count had a similar problem and every count phoned them," she said. She also said bundles of votes had been counted behind a curtain in her area because of strong guidance that no-one should see how the voting was going locally until the national result was known.
Last night a Welsh Office spokesman said its officials did not recollect giving any verbal guidance. The "no" camp were not satisfied, though.
The Conservatives' constitutional spokesman, Nigel Evans, said there should be an inquiry, possibly by the Welsh Select Committee of the House of Commons.
"This is an amazing inconsistency," he said. "If there have been massive irregularities that is appalling and of course it doesn't bode well for democracy."
Matthew Gunther-Bushell, strategist for the "Just say No" campaign in Wales, said there had been considerable confusion among both voters and electoral officers.
"I was astonished at the time, considering how slim the `yes' majority was, that the Government refused a recount which is normal in Parliamentary elections.
"It is too early to be talking about vote-rigging but this new evidence about the counting guidelines, added to the widespread concern over counting irregularities, makes the case for a recount overwhelming," he said.
Although people who wrote "yes" in the "no" box had their votes counted as "no," those who wrote "no" in the "yes" box had their papers spoiled. There is no clear evidence that this swayed the vote either way, but some activists believe it may have done.