The tests done on swabs from artifacts at the Diefenbaker Centre in Saskatoon were unable to come up with a definitive DNA match, George Dryden said Monday.
"This is yet another frustration for me," Dryden said in a statement.
"(But) I will never quit. I will discover the truth about my biological father."
Only one of several items tested yielded trace amounts of DNA, he said, but it was not a "clean sample" and it yielded no useful result.
Dryden, 42, of Toronto, also took a swipe at the University of Saskatchewan museum, suggesting it had not given the DNA company he had engaged to collect samples the full run of its collection.
The setback comes amid Dryden's quest to prove he was the only child of Canada's 13th prime minister, even though Diefenbaker is not known to have had any children.
Dryden believes his ailing mother, Mary Lou Dryden, who was a known confidante of Diefenbaker's, had a brief affair with the Conservative prime minister shortly after she married her husband, Gordon Dryden.
She has refused to confirm the affair. But Dryden did discover last June that Gordon Dryden, the man he had always believed to be his father, was in fact not a biological parent.
Dryden said he hoped other relatives of Diefenbaker's would allow DNA samples to be taken in an effort to prove his parentage — an issue he conceded has driven him a "little bit batty."
Initially, the museum dedicated to Diefenbaker said it would not allow the DNA testing because it didn't believe it had any usable material but later relented. The tests were carried out last month by a company from Thunder Bay, Ont.
Diefenbaker was Conservative prime minister from 1957 to 1963. He died in 1979.
Dryden said the media coverage of his story might have yielded an "exciting" new lead. He did not elaborate but said he hoped to have some answers within the next several weeks.