The presence or absence of gender differences in working memory, localized in the prefrontal cortex (PFC), has been debated in a few fMRI studies. However, the hypothesis of gender differences in PFC function has not been elaborated, and comparisons among hemodynamic parameters designed to test for gender differences are scarce. We utilized near-infrared spectroscopy during verbal N-back tasks on 26 male and 24 female healthy volunteers. Changes in the concentrations of oxy- (Delta[oxy-Hb]), deoxy- (Delta[deoxy-Hb]) and total hemoglobin (Delta[tot-Hb]) were recorded simultaneously. Delta[oxy-Hb] and Delta[tot-Hb] exhibited obvious gender differences, but Delta[deoxy-Hb] did not. Males showed bilateral activation with slight left-side dominance, whereas females showed left activation. The activation in males was more wide-spread and stronger than in females. Furthermore, females required a lower hemodynamic supply than males to obtain comparable performance, and only females exhibited positive correlations between hemodynamic parameters and behavioral performance. The results reinforce the existence of a gender effect in hemodynamic-based functional imaging studies. Our findings suggest that females possess more efficient hemodynamics in the PFC during working memory and emphasize the importance of studying the PFC to further a scientific understanding of gender differences.