The study addresses a longstanding debate about the pervasiveness of the disease in this ancient civilisation.
Researchers obtained mitochondrial genomic evidence of malaria, from the teeth of bodies buried in three Italian cemeteries, dating back to the Imperial period of the 1st to 3rd centuries Common Era.
The genomic data is important, because it serves as a key reference point for when and where the parasite existed in humans, and provides more information about the evolution of human disease, researchers said.
"There is extensive written evidence describing fevers that sound like malaria in ancient Greece and Rome, but the specific malaria species responsible is unknown," said Stephanie Marciniak, a former doctoral student in the Ancient DNA Centre.
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"Our data confirm that the species was likely Plasmodium falciparum, and that it affected people in different ecological and cultural environments," said Marciniak, now at Pennsylvania State University in the US.
"These results open up new questions to explore, particularly how widespread this parasite was, and what burden it placed upon communities in Imperial Roman Italy," she said.
Researchers mined tiny DNA fragments from dental pulp taken from the teeth.
They were able to extract, purify and enrich specifically for the Plasmodium species known to infect humans.
It was a difficult and painstaking process, complicated by the very nature of the disease.
Usable DNA is challenging to extract because the parasites primarily dwell within the bloodstream and organs, including the spleen and liver, which decompose and break down over time-in this instance, over the course of two millennia.
P falciparum remains the most prevalent malaria parasite in sub-Saharan Africa and the most-deadly anywhere, responsible for the largest number of malaria-related deaths globally.
The findings are published in the journal Current Biology.