Twenty‐eight olive cultivars (Olea europaea L.) from the World Olive Germplasm Collection of IFAPA in Cordoba were studied, analyzing the squalene concentration of their virgin olive oils (VOO). Squalene content ranged from 110 to 839 mg/100 g achieving a mean value for the set of olive cultivars of 502 mg/100 g. The high variability observed could be explained only by the genetic component. Five olive cultivar categories were established for the oil squalene concentration when a Hierachical discriminant analysis was performed. Virgin olive oil can be considered an important source of squalene, it may be used also for characterization and discrimination of monovarietal virgin olive oils. Practical applications: Squalene content in virgin olive oil has a genetic component. The variability for Squalene content between monovarietal oils can be used to choose the optimal VOO for nutrition studies. Squalene may be used to discriminate virgin olive oil of an olive cultivar from other monovarietal oils. Virgin Olive Oil squalene content show a significant genetic variability.