Rao, a member of Andhra Pradesh assembly and husband of union minister Daggubati Purandeswari, did not vote for any of the three candidates of the ruling Congress in the elections held Friday.
The EC, for the first time, added NOTA option in the ballot paper for RS polls.
The Election Commission, for the first time, added NOTA option in the ballot paper for Rajya Sabha polls.
Rao told reporters that he had conveyed his decision in advance to Chief Minister N. Kiran Kumar Reddy.
Claiming that his action was against 'injustice' meted out to Seemandhra, he said sentiments of Seemandhra people and their leaders were not taken into consideration while taking a decision on carving out a separate Telangana state.
Though two of the three Congress candidates were from Seemandhra, Rao decided not to vote for them either.
Another Congress legislator K. Kamla, who is also from Seemandhra, said she dropped a blank ballot paper in the ballot box.
Out of 276 members of assembly, 248 cast their votes. Seven candidates including a rebel of Congress are in fray for the six seats.