Tunisia is one of the world’s largest producers of olive oil, and it preserves pools of olive genetic diversity that are still unexplored. A recent prospection and collection program of the National Gene Bank of Tunisia (NGBT) focused on the vast oasis of Degache, in the south west part of Tunisia, where 47 samples were collected and genetically characterized through simple sequence repeat (SSR) markers. Identification and authentication of genotypes were obtained through comparison with reference cultivars belonging to the Olive National Collection of Tunisia (IOC) and with cultivars from Algeria, Italia, Syria and Lebanon. Degache olive genotypes showed large genetic variability, a significant diversity from the reference germplasm, and a clear differentiation from modern varieties. The population structure analysis identified four gene pools characterizing genotypes from different area of origin. Two gene pools appear to be more represented in germplasm from southern Tunisia, where environmental conditions at critical plant development phases, are harsher. This suggests that this germplasm might present traits of adaptation useful for breeding to improve resilience to abiotic stresses. Our results will support ex situ and in situ conservation activities of Tunisian olive germplasm pursued by the National Gene Bank of Tunisia.