Gregory Villemin was found tied up and drowned in the Vologne river in eastern France in October 1984, sparking a long and convoluted legal saga that transfixed France for years.
On Wednesday morning, police arrested an uncle of Gregory's father and the uncle's wife in the Vosges mountains.
The young victim's father's half sister was also detained in the same region, police sources told AFP. His grandparents were also interviewed.
The arrests "target people very close to the heart of this case and aim to clarify certain points and to provide answers to questions we have," local prosecutor Jean-Jacques Bosc said in a statement.
The case was reopened in 2010 to take advantage of new DNA techniques.
The death of "Little Gregory" as he became known led to one of France's most notorious post-war murder mysteries, as police sought to untangle a web of family hatred and local jealousies.
The day after Gregory's body was found, a letter arrived at the home of the child's parents -- who had been receiving anonymous hate mail since 1981 -- claiming responsibility for the murder and calling it "revenge".
Bernard Laroche, a cousin of the child's father, was charged with the murder a month after the boy's death, based on evidence given by a teenage sister-in-law.
He was released after she withdrew her claims, only to be shot dead in March 1985 by Gregory's father Jean-Marie Villemin who spent two and a half years in prison for the crime.
The dead boy's mother, Christine Villemin, was herself charged with the murder in 1985. But she was finally cleared eight years later and all charges against her were dropped.
The arrests on Wednesday were on charges of being an accomplice to murder, failing to denounce a crime and failing to help someone in danger, said local newspaper l'Est Republicain which first reported the story.
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