The girls were known to share a liver which would complicate the surgery.
Australian surgeons began operating early Friday to separate 15-month-old Bhutanese twins joined at the torso.
Nima and Dawa, and their mother Bhumchu Zangmo, arrived in Australia a month ago with the help of an Australian charity but doctors had delayed the surgery until Friday to ensure the twins were well-enough nourished to support the operation.
"We feel confident that we've put everything in place to move forward today," Joe Crameri, the head of paediatric surgery at Melbourne's Royal Children's Hospital, told reporters Friday.
He said the operation was expected to last around six hours and would involve 18 medical staff divided into two teams, one for each girl.
The girls were known to share a liver, but could also share part of the bowel, which would complicate the surgery, Crameri said, but added that the chance for a successful operation was "looking very positive".
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