Lesley Molseed, 11, was stabbed and sexually assaulted after leaving her home in Rochdale, Greater Manchester, on an errand in 1975.
Stefan Kiszko, a tax clerk from Rochdale, was convicted of her murder, but was freed on appeal in 1992 after serving 16 years in prison. He died shortly after.
Her body was found on moorland above the Oldham to Halifax road in Ripponden, West Yorkshire three days after she went missing.
A fresh appeal by West Yorkshire Police featured on BBC One's Crimewatch programme on Wednesday night.
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It may be that someone has harboured a suspicion for 27 years that a friend, relative or acquaintance could have killed this little girl
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Detective Chief Superintendent Max Mclean
Officers say they now have a DNA profile which was left by Lesley's killer.
New techniques helped to make the discovery from only a few fragments of evidence left from the crime in storage.
Detective Chief Superintendent Max Mclean is appealing for help in identifying the killer.
He said: "It may be that someone has harboured a suspicion for 27 years that a friend, relative or acquaintance could have killed this little girl.
"We now have the ability, through the development of scientific methods, to eliminate these people once and for all."
Lesley's mother, April, said: "When we heard the police had found DNA we were absolutely elated, it's like all our Christmases rolled into one - it gives us great hope."
On Lesley's disappearance she added: "After two hours, I knew she was gone - I could feel it in the whole of my body.
"And I never lost that feeling, it just grew stronger and stronger and I thought 'I'm never going to see her again'."