Har Gobind Khorana did not know when he was born. “The correct date of my birth is not known,” he wrote in 1968, continuing: “that [the date] shown on documents is January 9, 1922.” We do not know where that date came from. Though the family owned some ancestral land, his parents were poor, and Indians of that era and from that economic background rarely kept exact records of births or other such events. Not unexpectedly, no childhood photograph of Khorana is known to exist, and the few stories that are known about his childhood may well be apocryphal rather than a record of facts.

Khorana was born in Raipur, then a village of about a hundred people near the ancient city of Multan in Punjab in what is now Pakistan. The village was near what is now Kabirwala; otherwise, it has disappeared from maps though there are people still living in the area. None of Khorana's family remains there today. During the time of India's Partition in 1947 and the violent chaos that followed, they had all migrated to India, initially mainly to Delhi. Other than being Khorana's birthplace, nothing of significance has been recorded of Raipur.

According to his grandnephew, Alok Khorana: “family lore speaks of a mischievous child who liked to steal sugarcane from the sugarcane fields. Gobind described our ancestral home as consisting of a kitchen and bedrooms in one corner, with a courtyard housing cows and horses on the opposite end.” The family is believed to have owned that ancestral home for centuries. As far as can be told, the ancestral home does not survive. The family lost all their landed property during Partition as was typical for Hindus and Sikhs who fled Pakistan and Muslims who fled India.

Gobind's father, Ganpat Rai Khorana, was a patwari, that is, a village agricultural taxation clerk at the lowest level of the administrative system of British-ruled India. We know little about Ganpat Rai, except that he insisted on educating all his children. Khorana later recalled: “Although poor, my father was dedicated to educating his children and we were practically the only literate family in the village.”