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 about 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: 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 governments.
"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 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.
The Caucusus' "frozen conflicts"
Threw off Georgian rule in fighting in the early 1990s. A ceasefire was signed but the violence has threatened to reignite, especially since pro-Western Georgian President Mikhail Saakashvili was elected in 2004 and vowed to reunify the country. Russia has peacekeeping troops in South Ossetia.
A territory sandwiched between the Black Sea and the Caucasus mountains. It fought a 1992-3 war against Georgia in which it won de facto independence but no international recognition. It was isolated for years after its victory but it has then forged closer ties with Russia which has given Abkhaz residents passports and pensions.
It is part of Azerbaijan but has been controlled by Armenian separatists since armed conflict erupted in the 1990s. A major pipeline linking Caspian Sea oil fields to world markets passes a few kilometres from the conflict.
"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 Organisation for Security and Cooperation in Europe has said the vote will be neither free nor fair, and should not be recognised.
"The questions are loaded and suggestive, and from a one-sided perspective," said Claus Neukirch, a spokesman for Moldova's OSCE office.