An earthquake measuring magnitude 6.6 has struck central Italy near Norcia, 68 kilometres east-southeast of the city of Perugia according to the U.S. Geological Survey (USGS).
There were no immediate reports of casualties following the shock on Sunday.
According to the USGS, the earthquake struck at 7:40AM local Italian time and was 10 kilometres deep.
Central Italy has been on edge this week following Wednesday's two powerful earthquakes, measuring 6.1 and 5.5 on the Richter scale. These quakes left almost 5,000 people homeless and caused widespread damage but "miraculously" there were no fatalities.
The aftershocks from the earthquake may continue for weeks or months, according to the United States Geological Survey.
"We cannot rule out the possibility of similar sized or larger events, though the probability of a larger event is low," the USGS said in a statement.
Tremors from Sunday's earthquake were felt 135 kilometres away in the nation's capital of Rome.
It is Italy's largest earthquake since a magnitude 6.9 earthquake struck southern Italy in 1980. That quake killed almost 2,500 people and left 250,000 homeless.
The series of quakes has left buildings in the region unstable, with some structures on the verge of collapse, according to the Sydney Morning Herald.
The shock also comes two months after a 6.2 magnitude, 4-kilometre deep earthquake struck east of Rome on August 24, killing almost 300 people and devastating several towns.
Many of the structures that were destroyed in that quake had no anti-seismic protection, which led to Italian officials launching an investigation into the incidents.
The Italian civil protection department has begun checks on all towns affected by the morning's quake with all metro services in Rome suspended following the shock, according to the BBC.
Italy sits on two fault lines, making it one of Europe's most seismically active countries.