Research has identified a correlation between prenatal markers of testosterone (2D4D) and sport performance. This relationship is thought to be explained by several important psychophysiological variables such as physical fitness and mental toughness. The current study sought to add to this body of research by examining the differences between high and low 2D4D, in measures of gender identity (Bem Sex Role Inventory) and mental toughness (the 48-item version of the Mental Toughness Questionnaire). A sample of 116 recreational (n = 59) and competitive netballers (n = 57) completed the psychological measures and provided right-hand scans from which 2D4D ratio measures were drawn. The key results included a large effect of low digit ratio on emotional control, life control, and interpersonal confidence. These findings suggest that 2D4D could provide a marker for sporting potential and mental toughness in female sport participants. However, future research may wish to establish the relative contribution of prenatal factors (e.g., 2D4D) and socialization factors (e.g., involvement in a sporting context) on sporting ability and related psychological variables.