Police have said a DNA test carried out on a man in Cyprus has proved he is not Ben Needham, who disappeared in 1991.
Ben, from Sheffield, vanished when he was 21 months old while on holiday on the Greek island of Kos.
The man had spoken to officers after footage of him looking similar to how Ben might appear now was released.
A tweet from @FindBenNeedham, the account of the family's campaign, said it was "disappointing but they remain strong and resolute in search".
Ben's mother Kerry has always maintained her son is alive and was probably abducted.
"We are trying our best to keep strong and be there for each other and just keep waiting, however long it takes," Ms Needham added.
'Mound of earth'
She said last week the discovery of a blonde girl called Maria at a Greek Roma camp had given her family "fresh hope and maximum publicity".
Ben's family had spent his 24th birthday waiting for results of the DNA test.
He vanished on 24 July, 1991, after travelling to Kos with his mother and grandparents who were renovating a farmhouse in the village of Iraklise.
In 2012, a police operation focused on a mound of earth and rubble close to where Ben was last seen, but no trace of him was found.
A team of UK police officers spent a week searching the site close to the farmhouse.
His mother vowed to continue looking for him after the search failed to find anything.