Rajabbi Khurshed, 18, committed suicide 40 days after her arranged marriage to Zafar Pirov, 24.
She passed a government-required prenuptial exam but Pirov, from Tajikistan, demanded two further tests and called for a new wife after refusing to believe the results.
Pirov faces jail and stands accused of pushing his wife to commit suicide in the village of Chorbogh.
Ms Khurshed’s family claim Pirov cast their under-pressure daughter out and say she admitted she “couldn’t take it any longer” as she lay on her deathbed.
Her mother, Fazila Mirzoeva, told Radio Free Europe Khurshed had never had a boyfriend or intimate relationship before marriage.
Describing her daughter as a victim of “slander and violence” she has issued a plea to President Emomali Rahmon to intervene and save Khurshed’s reputation.
She said Khurshed dropped out of school to care for her two disabled brothers.
Pirov, who faces eight years in prison if found guilty, insists his wife was not a virgin.
He said: “My wife gave me a written statement that she allows me to get a second wife because she wasn’t a virgin when we got married.”
Medical checks before marriage were made compulsory for men and women in 2015 to prevent the spread of sexually transmitted diseases but expanded to include virignity tests.
Sex before marriage in Tajikistan is taboo and brides passing their virginity test receive a doctor’s letter to confirm the results.