There are reports of fatalities after Ukrainian national guardsmen opened fire on a crowd in the eastern town of Krasnoarmeisk.
The shooting took place outside Krasnoarmeisk town hall, where separatists had been attempting to hold an independence vote. Guardsmen had shut down voting hours earlier.
According to ITAR-Tass news agency, the number of deaths is as yet unspecified, however initial reports suggest at least one person had been killed and two more injured.
Earlier there were chaotic scenes during the referendums to determine whether to declare the two eastern Ukrainian regions of Donetsk and Luhansk as sovereign people's republics.
Polling has been beset by irregularities, with a severe shortage of polling booths, ballot papers being printed off unsupervised and CNN film crews claiming several people appeared to vote twice.
There have been heightened tensions at the polling stations after at least seven people were killed and others injured in the southern port city of Mariupol, in the Donetsk region on Friday (May 9).
Troops have been battling pro-Russian rebels, who have barricaded themselves in towns and cities in Donetsk and Luhansk.
In Donetsk, rubber bullets were fired in the air and a pro-Ukrainian head teacher said she had been threatened for not allowing separatists to use her school.
It has also been reported that police in Krasnoarmeysk had attempted to stop the voting.
The vote has been declared illegal by both the Kiev government and the international community and marks a further deepening of the political crisis in Ukraine.
Acting chief of the Ukrainian presidential administration Sergiy Pashinski said: "The turnout throughout the whole region amounts to zero as far as formalities are concerned. I would like to underline one more time it's not a referendum. It is a desultory attempt by terrorists, bandits and killers to cover their activity by having citizens vote in Luhansk and Donetsk."
A Foreign Office spokesman said: "It is regrettable that separatists who are causing so much disruption to the lives of ordinary citizens are going ahead with their illegitimate, so-called referendum today.
"All Ukrainians will have a proper opportunity to express their democratic choice in the forthcoming presidential elections on May 25. The focus must be on ensuring that free and fair elections can go ahead in a calm and stable environment."
Meanwhile, France and Germany called on Russia to prove it had withdrawn its forces from the Ukrainian border.
German Chancellor Angela Merkel said the referendum would be viewed as illegitimate as both she and French President Hollande called for 40,000 Russian troops to be pulled back from the border.
Earlier this week, Russian President Vladimir Putin said his country's forces had moved back to training bases, although the White House and Nato both said they had seen no movement.
It is feared the unrest could descend into civil war.
The referendum has been held in defiance of Moscow by pro-Russian militants. Polls close at 8pm UK time.