Preliminary results from the plebiscite were expected shortly.
Pre-poll surveys suggested Morales would easily survive the vote, but that up to three of the eight governors on the ballot might lose their jobs. Only two of the governors are pro-Morales.
"My big dream is to have unity among the Bolivian people," Morales said as he cast his ballot in Villa 14 de Septiembre, a town in the central Chapare state that is a bastion of support for him.
The former coca farmer who became Bolivia's first indigenous leader in 2006 called the referendum in a bid to get the upper hand over opposition conservative governors resisting his socialist reforms.
He was banking on continued massive support among Bolivia's indigenous majority, of which he is part, to hold on to his post.
In the December 2005 election that brought him to power, he won 53.74 percent of the ballots.
Since then, though, opposition to him has hardened as he imposed a program of nationalizations and sought to rewrite the constitution to benefit indigenous folk.
Four opposition governors in Bolivia's lowlands -- an area rich in gas fields and farmland that provides most of the country's wealth and which is run by an elite of European descent -- have countered with pushes for autonomy. Morales has dismissed that campaign as "illegal".
The ensuing crisis has deeply divided the country along ethnic, political and regional lines.
Morales has been prevented from landing his plane in opposition states because of sometimes violent protests, and demonstrations against his reforms have dogged his administration.
Analysts said the referendum would likely change little, and could even harden the political conflict.
"Win or lose, Evo seems to have settled in as the revered leader of half the country, and the devil in disguise for the other half. That presents a real problem for governing," said Jim Shultz, the US director of the Democracy Center in Bolivia.
Differing official interpretations over how many votes were needed to topple the governors raised the prospect of disputes in the referendum's wake.
According to congress, the governors can be ousted if the number of "no" votes exceed the amount of support they received in the 2005 elections, which ranged between 38 to 48 percent of ballots.
But the National Electoral Court has said the proportion needed to bring down a governor is 50 percent plus one ballot.
That could create a scenario in which Morales insists that a governor has been ousted according to congress, but the governor refuses to go, citing the court's standard.
All the governors advocating autonomy are expected to be comfortably confirmed.
One of them, Ruben Costas, the governor in charge of Santa Cruz state, smiled as he voted.
"I am feeling good, happy, because this is going to let us accelerate even more our process for autonomy," he said.