Kendra and Maliyah Herrin were born fused at the midsection, sharing a liver, kidney, pelvis, one set of legs and part of their intestines.
Surgeons gave each girl one leg, split their liver and intestines and reconstructed their bladders and pelvic rings.
Kendra kept their one functioning kidney, while Maliyah was prepped for kidney dialysis and a transplant in three to six months.
The Herrins' separation is believed to be the first performed on conjoined twins with a shared kidney, said the hospital's chief of paediatric surgery, Dr Rebecka Meyers.
Meyers said the surgery, while complicated, had not presented any major surprises for the team of six surgeons, two anaesthesiologists, two urologists, one radiologist and more than 25 nurses and medical technicians.
But the days ahead will still be tough, she said.
"We have big concerns with both girls," said Meyers. "Maliyah's big concern is her kidney function and her dialysis and Kendra's big concern is going to be the coverage of her abdomen."
Surgeons said Kendra has less muscle and tissue for use in closing her abdomen.
They planned to use synthetic skin to help solve the problem.
Meyers said Maliyah did not require immediate placement on dialysis, but she expects that may happen within a few days.
When the girl is strong enough, she will get a kidney from her mother, Erin.
The twins are expected to remain in intensive care for about a week and recover in the hospital for at least a month before doctors can consider sending them home.
Conjoined twins occur about once in every 50,000 to 100,000 births.
Only around 20 per cent survive to become viable candidates for separation.
In most instances, conjoined twins undergo separation surgery between ages six and 12 months, but the girls' shared kidney forced a delay.
Erin Herrin, 25, broke into tears and fell into the arms of her 26-year-old husband, Jake, on learning of the successful surgery.
The couple, who also have a six-year-old daughter and twin 14-month-old boys, said they were eager to tell the girls how brave they had been and how proud they have made the family.
Jake Herrin even cracked a few surgery jokes.
"Most people say, 'You made it through in one piece," he said. "We can say, "You guys made it through in two."'