The investigation in Carini -- a small town near Palermo -- centers on the castle where Baroness Laura Lanza was killed in 1563 with her lover Ludovico Vernagallo when they were caught in bed together.
"Justice wasn't done back then," said Gaetano La Fata, Mayor of Carini, who has decided to reopen the case and exhume the remains of the lovers.
"We hope that DNA tests and criminal profiling will help us discover the motive behind the crime and establish whether there was more than one assassin," he told Reuters.
The Baroness's father Cesare confessed to the honor killing in a letter to the king, which is currently archived in the Chiesa Madre church in Carini.
"Legend has it, however, that Cesare Lanza did not act alone, but was helped by his son-in-law, Don Vincenzo La Grua," said the Mayor.
Rumours passed down through generations of Sicilians have it that the husband was motivated by plans to marry again. La Grua may also have feared his rival, Vernagallo, would attempt to claim financial rights for fathering children with his wife.
In reopening the 'cold case', La Fata has asked the local police to work together with the ICAA (International Crime Analysis Association) headed by Marco Strano, psychologist and criminologist for the Italian State Police.
"The idea for the investigation began as a joke," Strano told Reuters. "I visited Carini in June and when I met La Fata I teased him for not having resolved the murder yet, so he challenged me to solve it."
"There was a trial held at the time, but though both father and son-in-law had their properties temporarily confiscated, they were soon declared innocent, probably thanks to their noble status and the legal right for fathers and husbands of adulterous women to commit honor killings," said Strano.
It is thought the two lovers are buried in a common grave under the crypt of the Chiesa Madre church in Carini.
"If we are lucky enough to find and identify their bones, it might be possible to verify the cause of death, whether they were run through with a sword or stabbed with a dagger. If there was more than one weapon used, it's likely there was more than one murderer."
The team of crime analysts, made up of American and Italian experts in forensic science and criminal pathology, are in the process of making a 3D computer model of the 11th century castle, including the room overlooking the Gulf of Carini where the murder took place.
"We hope to map the killer's path from the courtyard to the crime scene, and work out whether it's likely there were servants in that part of the building at the time who might have seen the murderer or an accomplice," Strano said.
The investigation coincides with a project to rebuild parts of Carini Castle that have collapsed over time. The crime scene has recently been restored. A red handprint has been painted on the wall to mark the spot where -- legend has it -- the struggling Baroness left a bloody imprint, which reappears every year to mark the anniversary of her murder.
Mayor La Fata hopes that the project will help unravel some of the mystery that surrounds the lovers, whose story continues to intrigue visitors and locals alike.
"Several years ago we tested areas of the castle we knew the Baroness lived in with electromagnetic field meters, and the results were very strange," La Fata said. "In certain rooms it was as if there were ghosts in the castle, as if the murdered Baroness lives on."
Reporting by Ella Ide; Editing by Stephen Brown and Paul Casciato