Vanessa Trump, an ex-model, is seeking an uncontested divorce in a New York court.
The couple, both 40, have been married since 2005. They have five children.
Mr Trump Jr played a prominent role in his father's election campaign.
He stayed in New York after the election and now co-runs the family business with his brother Eric Trump.
He has become the subject of an investigation over possible collusion between the presidential campaign and Russia in 2016.
President Trump denies any collusion. Donald Trump Jr arranged a meeting between a group of Russians and campaign officials after being told of possible damaging information about Mr Trump's Democratic challenger Hillary Clinton.
An uncontested divorce means that custody over the couple's children and assets is not disputed during legal proceedings.
Page Six, the New York Post gossip column that broke the story, carried a statement from the couple saying: "After 12 years of marriage, we have decided to go our separate ways.
We ask for your privacy during this time."
Earlier this year, reports swirled that the couple had marital problems, pointing out to Mr Trump Jr's frequent travel.
Last month, Vanessa Trump and her mother were taken to hospital as a precaution when she opened a letter addressed to her husband containing white powder.