The New York City-to-Ohio Greyhound Charter bus crashed at about 1:30 a.m. in the westbound lanes of I-80 in Union County's White Deer Township, near the exit for Williamsport.
Investigators say it slammed into the back of a tractor trailer.
One woman was killed and at least 44 others were taken to five area hospitals. Officials say there were 49 passengers on board, as well as the driver.
Police said several people had to be airlifted.
Greyhound spokeswoman Alexandra Pedrini said 38 of the 49 passengers were treated and released by late Wednesday afternoon, and then placed on replacement buses to their final destination. She said the bus driver, Sabrina Anderson, 42, was one of four people in critical condition at Geisinger Medical Center in Danville.
The hospital is treating 13 patients, two others are listed as serious, and seven others are listed as fair. Four patients were treated and released.
Officials at Evangelical Hospital in Lewisburg say they are treating 14 patients.
Relief busses were sent from Philadelphia to pick up uninjured passengers and passengers who had been treated and released from the hospital.
The driver of the tractor-trailer, Akos Gubica, 36, of New Jersey, told The (Sunbury) Daily Item that his rig was struck from behind by the bus.
Greyhound declined to comment on that, saying the company was cooperating with authorities in their investigation and was conducting an internal probe.
The driver has been with the company for more than 12 years