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Critics of Sunday's vote fear it could set a precedent for other regions involved in "frozen conflicts" seeking independence from ex-Soviet States.
Moldova fought separatists in Trans-Dniestr in 1992 in a war that left around 1,500 people dead and has said it will not recognise the vote.
The conflict was ended by Russian troops who have remained in the tiny region, currently numbering 1,200 and Moscow has sent a delegation to observe the vote.
The region's 390,000 voters are being asked two questions are asked two questions: whether they back independence and attachment to Russia or whether they reject independence and want to integrate with Moldova.
Igor Smirnov, Trans Dniestr's president defended the referendum which is not recognised by any western goverments.
"I voted for statehood, for strategic partnership with Russia ... I think all our Dnestr people will vote the same way," he said after casting his ballot in Tiraspol, Trans-Dniestr's main town.
Voters had until 8pm local time to cast their ballots and were offered cheap food and drink when they did so with the red and green separatist flag adorning Tiraspol's streets.
"I was an orphan and was raised by a Russian soldier," Galina, 62, said by her polling station. "Russia is our only choice. I voted for it and I'm delighted."
Moldova accuses Russia of abetting the separatist rebels and it is the same story in Georgia where the breakaway region of South Ossetia is looking to hold a similar referendum in November.
Both Trans-Dniestr and South Ossetia have sought precedents in the former Yugoslavia, where voters in Montenegro have opted for independence and talks in Kosovo are likely to lead to a similar result.
"We are trying to duplicate the experience from Kosovo," Valery Litskai, Dnestr's self-styled foreign minister said on the eve of the vote. "We are looking at them and they are looking at us."
The rest of tiny Moldova, on the opposite side of the Dnestr River, is ignoring the vote and local media said the president, Vladimir Voronin, told officials at a meeting on Saturday to remain calm.
The Organization for Security and Cooperation in Europe has said the vote will be neither free nor fair, and should not be recognized.
"The questions are loaded and suggestive, and from a one-sided perspective," said Claus Neukirch, a spokesman for Moldova's OSCE office.
/Aljazeera.Net/