According to Greece’s public broadcaster, there are between 50 and 60 people missing.
Many of the victims were thought to be university students returning home late on Tuesday after a long holiday weekend.
Officials said the death toll was expected to rise further — temperatures in one carriage had risen to 1300C after it caught fire.
At least 130 people were injured, with seven suffering severe burns.
Flags flew at half-staff in Athens and in Brussels and the Greek government declared three days of national mourning.
As a result of the disaster, Greece’s Minister for Infrastructure and Transport, Kostas Karamanlis, has submitted his resignation, saying he was taking responsibility for the state's "long-standing failures" to fix a railway system he said was not fit for the 21st century..
“I consider citizens having trust in the political system a necessary element of our democracy,” he said.
“This is called political responsibility.
“For this reason, I submit my resignation as minister.”
Karamanlis said he felt it was the least he could do to as a sign of respect to the victims.
Authorities are working to establish how the high-speed passenger train collided with another carrying shipping containers, coming in the opposite direction and on the same track at speeds thought to be up to 160 kilometres per hour.
The local station master, in charge of signalling, has been charged with causing mass deaths through negligence and causing grievous bodily harm through negligence, a police official said.
The 59-year-old man denied any responsibility, attributing the accident to a possible technical failure, the official said.
Yiannis Ditsas, head of the railway workers union, told Skai TV that automatic signalling at the site of the crash had not been working. There was no immediate official comment.
The passenger train was carrying 342 travellers and 10 crew, with two crew on the cargo train, Hellenic Train data showed.
In Larissa, where many victims of the crash had been taken, Nikos Makris sat on a pavement outside the hospital.
His wife's sister was travelling in one of the first two carriages.
"She is missing. We have been waiting here since 2am," he told reporters.
"Now we are waiting to do a DNA test. We will be lucky to have a body to bury," he said.
Others were angry. The relative of one victim shouted: "Some bastard has to pay for this."
Greece sold railway operator TRAINOSE to Italy's Ferrovie dello Stato Italiane in 2017 as part of its international bailout program, expecting hundreds of millions of euros to be invested in rail infrastructure in the coming years.
The Italian division maintained responsibility for passenger and freight, while the Greek state controlled infrastructure.
"Everything in this tragedy points, unfortunately, mainly to human error," Greek Prime Minister Kyriakos Mitsotakis said in a televised address on Wednesday.
In later statements he said he had accepted the resignations of senior officials in rail operator OSE and its subsidiary ERGOSE.
In Athens, about 1000 people protested outside the offices of Hellenic Train, another branch of the rail network, where some hurled stones at windows.
ANSA
