"An unauthorized use at the Republican Convention against our wishes," the British band said in a succinct statement posted on Twitter.
Trump -- who took the stage to "We Are the Champions" through a cloud of dry ice on the Cleveland convention's first day Monday -- went ahead with the song despite public pleas from the band.
Guitarist Brian May said in June that Trump had not asked before previously playing the song and that the band would "never give permission."
"Our music embodies our own dreams and beliefs, but it is for all who care to listen and enjoy," May wrote on his website at the time.
Adam Lambert, the US singer who has been performing with Queen on tour, was more outspoken and noted that "We Are the Champions" was written by Queen's Freddie Mercury, one of the most prominent musicians to die from complications of AIDS.
Mercury, born in what is now Tanzania to an Indian family which was forced to flee to England when he was a teenager, has become a gay icon but was largely quiet about his sexuality in public.
The row with Queen is only the latest between Trump and musicians.
Adele, the Rolling Stones, REM and Neil Young have all asked the billionaire right-wing populist to stop playing their music at campaign rallies.
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