The prefrontal cortex (PFC) is thought to play an important role in "higher" brain functions such as personality and emotion that may associated with several gender-related mental disorders. In this study, the gender effects of functional connectivity, cortical lateralization and significantly differences in the PFC were investigated by using resting-state functional optical tomography (fOT) measurement. A total of forty subjects including twenty healthy male and twenty healthy female adults were recruited for this study. In the results, the hemoglobin responses are higher in the male group. Additionally, male group exhibited the stronger connectivity in the PFC regions. In the result of lateralization, leftward dominant was observed in the male group but bilateral dominance in the female group. Finally, the 11 channels of the inferior PFC regions (corresponding to the region of Brodmann area 45) are significant different with spectrum analysis. Our findings suggest that the resting-state fOT method can provide high potential to apply to clinical neuroscience for several gender-related mental disorders diagnosis.