The president should not be "afraid" to choose his heir and his word would be "final", Grace Mugabe said.
Mr Mugabe is the world's oldest ruler, and his Zanu-PF party has nominated him to stand for re-election next year.
But rival factions have been vying to strengthen their position as concern about his health mounts.
Mr Mugabe looks increasingly frail, and has been on three medical trips to Singapore this year.
However, his aides have played down fears about his health, saying he goes to Singapore only for specialised eye treatment.
"President, don't be afraid. Tell us who is your choice, which horse we should back," Mrs Mugabe told a meeting of  Zanu-PF's women's wing, AFP news agency reports.
"If you tell us the horse to back, we will rise in our numbers and openly support that horse. Why should our horse be concealed?" she added, in a speech switching between English and local language Shona.
Mr Mugabe was present when his 52-year-old wife made the comments, but did not respond.
She has been rumoured as a potential successor, as has Vice-President Emmerson Mnangagwa.
Mr Mugabe has ruled Zimbabwe since independence in 1980, and is expected to defeat his main rival, Morgan Tsvangirai, in next year's poll.
The opposition says the electoral commission is biased and will rig the poll in Mr Mugabe's favour.
Last year, Zanu-PF youth leader Kudzai Chipanga said that "it was written in the Bible that he [Mr Mugabe] won't just rule the country but would die in office".
In February, Mrs Mugabe said her husband was so popular that if he died, he could run as a corpse and still win in an election.
Zimbabwe's President Robert Mugabe, 93, has been urged by his wife to name his successor in order to end divisions over who the next leader will be.