The operation started at HCM City-based Children Hospital 2 at 06.00am and is expected to last 12 hours.
The twins, from Ninh Thuan province, were born 14 months ago and they had since undergone a special care mechanism at the hospital before the surgery.
Dr Truong Quang Dinh, vice director of the hospital, said one of the twins was in good health condition, and the other was suffering after-birth cerebral haemorrhage and pneumonia with respirator support.
Doctors decided to separate the twins because any deterioration of the weaker baby can threat the lives of the two, Dinh said.
In December 2012 HCM City Children Hospital 2 doctors successfully separated twins joined at the abdomen and chest.