The quake, which measured 6.6 according to the US Geological Survey, was bigger than the one on 24 August that killed almost 300 people.
Many people have fled the area since then, helping to avoid a new devastating death toll.
With thousands already made homeless, a leading seismologist warned that the earthquakes could go on for weeks in a domino effect along the central Apennine fault system.
The latest quake was felt across much of Italy, striking at 7.40 a.m. (0640 GMT), its epicentre close to the historic Umbrian walled town of Norcia, some 100 km (60 miles) from the university city of Perugia.
Panicked Norcia residents rushed into the streets and the town's ancient Basilica of St. Benedict collapsed, leaving just the facade standing.
Nuns, monks and locals sank to their knees in the main square in silent prayer before the shattered church.
"This is a tragedy. It is a coup de grace. The basilica is devastated," Bishop Renato Boccardo of Norcia told Reuters.
"Everyone has been suspended in a never-ending state of fear and stress. They are at their wits' end," said Boccardo, referring to the thousands of tremors that have rattled the area since August, including two serious quakes on Wednesday.
