The bus slammed into the back of a tractor-trailer hauling garbage on Interstate 80 near Lewisburg, Penn., at about 1:40 a.m., with 50 passengers on board.
A spokeswoman for Greyhound said that at least 45 people, including the bus driver, were taken to five area hospitals, with 20 ambulances from five counties responding to the crash scene. Four of the passengers are listed in critical condition.
By late Wednesday morning, four of the patients were treated and released.
The driver of the bus, who has been driving for Greyhound for 12 years, will be placed on temporary leave as the bus line investigates the crash. The driver was not identified by name.
A New Jersey man that identified himself as the driver of the tractor-trailer said that he was traveling the speed limit when he was rammed from behind by the bus.
36-year-old Akos Gubica said he did not know why the bus struck his tractor-trailer. He was not injured in the accident.
Pennsylvania State Trooper Matthew Burros said investigators were recreating the crash scene Wednesday morning.