The Portuguese forward – who plays for Juventus in Italy's Serie A league – has vehemently denied the accusations, and his lawyer Peter Christiansen said the request was standard procedure, AFP reported.
"Mr. Ronaldo has always maintained, as he does today, that what occurred in Las Vegas in 2009 was consensual in nature, so it is not surprising that DNA would be present, nor that the police would make this very standard request as part of their investigation," he said in a statement.
The Las Vegas Metropolitan Police Department said it had made an official request to Italian authorities, adding it is "taking the same steps in this case as in any other sexual assault to facilitate the collection of DNA evidence".
Former model Kathryn Mayorga, 34, of Las Vegas, made the accusations against the now 33-year-old Ronaldo in a complaint filed last year in the state of Nevada.
She claims after meeting the footballer at a Las Vegas nightclub, he raped her at his hotel suite on June 13, 2009 – just before he joined Real Madrid from Manchester United for a then-world-record €94 million ($108 million).
Her lawyers have said previously she immediately reported the alleged rape to the Las Vegas police and underwent a medical examination.
A private mediation was organized with representatives of Ronaldo, Mayorga and their lawyers, where she alleges she was paid $375,000 to keep quiet.
But Ronaldo's lawyers have said that the non-disclosure agreement signed with the former model was "by no means a confession of guilt".
Mayorga's attorney has previously said the model agreed to an out-of-court settlement to keep her name from going public, but was inspired to speak out by the #MeToo movement against sexual harassment.
Her attorney could not immediately be reached for comment, but in the past said the agreement should be made null and void because it was made when she was suffering from extreme psychological duress.
Ronaldo is widely considered one of the greatest footballers in the history of the game, having won the Golden Boot award – given to the leading goal scorer in any European league – a record four times.
Juventus signed him last year for €100 million ($115 million), the most ever paid for a player aged over 30.
Ronaldo has long been marketed as a world face of football and his image has become a global money spinner. When the rape allegations first came to light, shares in Juventus dipped on the stock exchange.