Netrebko said her gift to the Donetsk opera and ballet theatre was "a step to support art where it is needed now".
Russian Channel 5 TV showed her giving the cheque to Oleg Tsarev, a leader of the armed separatists in Donetsk.
Russian government support for the rebels has been denounced by the West.
The famous soprano made her donation in St Petersburg, where she is a star of the Mariinsky Theatre. She said performers in Donetsk were struggling on with their art despite the freezing cold.
Other top names in Russian culture have also voiced support for President Vladimir Putin's stance on Ukraine, notably the government's annexation of Crimea and support for the pro-Russian separatists in Donetsk and Luhansk.
The Russian celebrities backing the Kremlin over Ukraine include variety singer Iosif Kobzon, film director Nikita Mikhalkov, conductor Valery Gergiev and viola virtuoso Yuri Bashmet.
Internationally renowned Russian opera singer Anna Netrebko has donated 1m roubles (£12,000; $19,000) to a theatre in rebel-held eastern Ukraine and posed with a rebel flag.