Both victims had been in a 2002 Ford Ranger that crashed in the southbound lanes, south of state Route 54.
The sisters got out of the pickup, which was blocking traffic lanes, and were hit by an oncoming 2009 Dodge Caliber driven by a 44-year-old woman, CHP officers said.
The women were identified as 27-year-old Monica Moreno and her 20-year-old sister Veronica Moreno. Both were residents of Chula Vista.
The crash was reported shortly after 1:30 a.m., according to the California Highway Patrol.
The crash left the southbound lanes blocked in the area, and the connector ramp from eastbound state Route 54 to southbound I-805 closed, according to Caltrans and the CHP. A SigAlert was issued, but the lanes were cleared and the alert lifted shortly after 5 a.m.