Jacob’s Well, located in modern city of Nablus and ancient Shechem (Tall Balata) in the northern West Bank of Palestine, attracts modern day tourists and pilgrims. It is found in the eastern suburbs of the city. Since 333 AD, pilgrims have been writing accounts of the well, and it has been venerated by both Christian and Jewish communities throughout its history. It is believed to be the well referred to in the New Testament, where Jesus conversed with a Samaritan woman, the orthodox saint, Photini. It now forms the central feature in the crypt of the St Photini Greek Orthodox church in the walled grounds of a monastery. In order to gain more information on the chronology of the site, we analyzed human skeletal remains found at the site in 1997. These consist of three skulls and a femur. One of the skulls was found in a sarcophagus alongside the church and the two other skulls and a femur were found in a burial ground alongside the monastery, north of the church, over which a room has now been built. Radiocarbon analysis reveals that the remains date to four historical periods or events: the early Christian period, before structural additions to the well by Constantine the Great in the fourth century; the Samaritan Revolts (AD 529 and 556), the Sassanid Invasion (AD 614–628), and Abbasid rule (AD 750–1258). Dating of one skull suggests it may have been that of Germanus, a fourth century bishop of Nablus, and that there may have been a very early structure, shrine, or burial chamber at the site before the fourth century. We provide contextual information based on historical and contemporary literature.