History has well documented the execution of Queen Ketevan of Georgia by the Persian Emperor of modern day Iran. Based on historical records, in 1624 two Augustinian friars unearthed queens remains and one of them brought the relic to St. Augustine convent Goa, India. We carried out ancient DNA analysis on the human bone remains excavated from St. Augustine convent by sequencing and genotyping of the mitochondrial DNA. The investigations of the remains revealed a unique mtDNA haplogroup U1b, which is absent in India, but present in Georgia and surrounding regions. Since our genetic analysis corroborates archaeological and literary evidence, it is likely that the excavated bone belongs to Queen Ketevan of Georgia.