Thomas Kwoyelo, commander of the Lord's Resistance Army rebels, was charged before the International Crimes Division court in the northern town of Gulu with 53 counts of wilful killing, hostage taking, destruction of property and causing injury.
The 39-year-old Kwoyelo, who was a "colonel" under the command of the elusive LRA supremo Joseph Kony, denied the accusations.
Kwoyelo was captured in March 2009 in Garamba forest in the Democratic Republic of Congo during a sweep by regional armies against the LRA rebels who had fled from northern Uganda.
"All attacks were either committed by him or were carried out with his full knowledge," according to the charge sheet. His hair neatly combed and wearing a green shirt, Kwoyelo shook his head as the charges were read out in the packed coutroom.
At the opening of the trial, Uganda's main judge Yorokamu Bamwiine said: "Ugandans and the international community have a feeling that justice must be done and it is there to be done."
"This is regardless of who wins or who loses," he added.
Kwoyelo is not among the top LRA commanders, including Kony wanted by the International Criminal Court for rape, mutilation, murdering and forcefully recruiting child soldiers.
Tens of thousands of people were killed and nearly two million displaced in the two decades of fighting between Kony's LRA and the Ugandan government.
Uganda's civil war effectively ended in 2006 when a peace process was launched but Kony and his top commanders have remained elusive and continued to commit atrocities in remote areas of neighbouring countries.
The LRA supremo, a semi-literate former altar boy, took charge in 1988 of a regional rebellion among northern Uganda's ethnic Acholi minority.