The child who had been taken to Christiana Hospital in Delaware died about 1 a.m., state police spokeswoman Linette Quinn said.
The girl was one of seven wounded in the school shooting Monday, the nation's third in less than a week, in a bucolic area of Lancaster County. Three girls, and the gunman, died at the school, authorities said.
Quinn said the two girls who died in hospitals early Tuesday had suffered "very severe injuries, but the other ones are coming along very well."
Another victim, a 7-year-old girl, died about 4:30 a.m. at Penn State Children's Hospital in Hershey, hospital spokeswoman Amy Buehler Stranges said.
"Her parents were with her," Buehler Stranges said. "She was taken off life support and she passed away shortly after."
A 6-year-old girl remained in critical condition and a 13-year-old girl was in serious condition at Penn State Childrens, Buehler Stranges said. She said the names of the children were not being released.
Charles Carl Roberts IV, apparently spurred by a grudge two decades old, wrote his wife what authorities described as suicide notes, took guns and ammunition and went to a nearby one-room schoolhouse, where he killed three girls, critically injured seven more, and took his own life, authorities said.
Roberts was armed with a loaded 12-gauge shotgun, a 9 millimeter shotgun, a stun gun, plastic handcuffs, enough two-by-fours to board up all the exits, six rounds of ammunition and a change of clothes, reports CBS News national correspondent Byron Pitts.
"I don't believe there was any sort of malice toward the Amish necessarily," State police Commissioner Jeffrey B. Miller said on CBS News' The Early Show. "I think he sought out a target of opportunity where he had female victims that were young in age, between the ages of 6 and 13, and I think that was really the focus, a place he could get into and out of."
Roberts, 32, of Bart, was not Amish, did not appear to be targeting the Amish and apparently chose the school because he was bent on killing young girls as a way of "acting out in revenge for something that happened 20 years ago," Miller said Monday, adding Tuesday morning, "He would have been 12 years old."
Roberts, now a father of three, also had a daughter who died as an infant.
Two young students were killed at the West Nickel Mines Amish School, along with a female teacher's aide who was slightly older than the students. Seven others, most shot at point-blank range, were taken to hospitals, authorities said.
"When I arrived at the scene, many of our troopers were covered in blood from ... bringing children out and being with them," Miller told Early Show co-anchor Harry Smith. "The one trooper, you know, the poor little girl had part of her head blown off by a round, and he held her in his arms and was with her while she was still alive for almost an hour, until she passed."
According to investigators, Roberts had finished his milk deliveries before dawn Monday, dropped his own children off at their school bus stop, then at about 10 a.m. pulled up at the Amish school, which had about 25 to 30 students ranging in age from six to 13.
The small one-room schoolhouse is the size of a two-car garage, reports Pitts.