Long cited by authorities as his final resting place, there had been rival claims - but now arguments over the nationality of 15th-century explorer are also set to be laid to rest.
A divisive figure, Columbus led Spanish-funded expeditions from the 1490s onward, opening the way for the European conquest of the Americas.
Researchers, led by forensic expert Miguel Lorente, have been testing tiny samples of remains buried in the cathedral, comparing them with those of his known relatives and descendants.
The findings are due to be announced in a documentary titled Columbus DNA: The True Origin on Spain's national broadcaster TVE on Saturday.
Briefing reporters on the research on Thursday, Lorente did not reveal the conclusions, but said they had confirmed previous theories that the remains in Seville belonged to Columbus.
"Today it has been possible to verify it with new technologies, so that the previous partial theory that the remains of Seville belong to Christopher Columbus has been definitively confirmed," he said.