The incident happened around 8 a.m., while the child's mother was driving a Chevrolet silver sedan northbound along the 55 Freeway in Orange, California Highway Patrol said.
The boy was sitting in a booster seat in the right rear passenger seat when he was shot during "some type of road rage incident," California Highway Patrol Officer Florentino Olivera said during a press briefing. He did not know how many shots were fired. The mother was not hurt, he said.
The child was pronounced dead at a nearby hospital. He was identified by family as Aiden Leos, a kindergartener at Calvary Chapel Yorba Linda. He was being driven to school by his mother when the shooting happened.
"She (Aiden's mother) was merging to the right side to get away from this person, and as you can see if you go online and look at the photos, there's one bullet shot in the trunk that went through the trunk and right through my nephew," his uncle, John J. Cloonan III, said at a press conference Friday evening.
Aiden had turned 6 just last week, his family said.
The suspected shooter remains at large and officers are looking for a white sedan, Olivera said.
Cloonan said the boy's mother saw a woman driving the vehicle and a man in the passenger seat, but was not sure who fired the shot.
Highway patrol officers are asking the public for help, including anyone driving on the freeway this morning between 7:55 a.m. and 8:15 a.m.
"Please help us find the people that did this to my little brother," his sister, Alexis Cloonan, said at the press conference. "He's only 6 and he was so sweet. ... I loved having him in my life."
"If your vehicle has a dashcam, we're asking you, please call our CHP office in Santa Ana, provide that video," Olivera said. "If you were driving by, you saw something that was not right, call it in -- even if it wasn't something major -- call our office. We want to hear from witnesses."
At least 80 incidents of cars being shot at while traveling on Southern California freeways have been reported since late April, ABC News Los Angeles station KABC reported on Thursday. Most were during the morning commute and there were no significant injuries, the station reported.
Friday's deadly shooting is isolated and unrelated to recent window-shattering incidents along Southern California freeways, Olivera said.
ABC News' Sarah Hermina contributed to this report.