The San Bernardino County Sheriff's Department said the car crashed into the water in the 13000 block of Main Street at about 6:45 p.m.
The car careened off the road, crashed through a guardrail and went upside down into the aqueduct.
Authorities were able to pull two children from the water as well as a deceased adult. They had searched for hours for a third child, but he has not been found.
HESPERIA UPDATE: 2 confirmed dead in aqueduct crash, believed to mother & child; 1 child at hospital, 3rd child still missing in water. pic.twitter.com/TlqFeTLeQL— Marc Cota-Robles (@abc7marccr) March 3, 2017
Dive teams said the water was very cold, there was bad visibility and the currents were strong.
The children were identified as Noah Abbott, 2, who is still missing, Jeremiah Abbott, 3, who died at the hospital, and Elijah Abbott, 10, who remains hospitalized.
Their mother, Cristina Estrada, 31, was pronounced dead at the scene.
The cause of the crash remains under investigation. Crews shut down Main Street and Escondido Avenue in the area.
A GoFundMe account has been set up to pay for funeral and medical expenses the family will incur. You can find it at gofundme.com/christina-jeremiah-noah-abbotts.