In this article, we analyzed the concentration of coumarins in leaves of female and hermaphrodite individuals of the gynodioecious shrub Daphne laureola, along an elevational gradient in southern Spain. Combining HPLC and NMR techniques, we identified three different glycosides of 7-methoxycoumarin in leaves of this species. Total coumarin concentration averaged between 60 and 120 mg/g dry weight for mature summer leaves of D. laureola growing at six different populations. As predicted by optimal theory, females tended to have a higher concentration of coumarins than hermaphrodites, thus upholding the idea that male reproductive function is costly for hermaphrodites. Furthermore, concentrations in females but not hermaphrodites were positively correlated with increasing population altitude, and the magnitude of gender divergence in coumarin concentration varied among populations, suggesting that the cost of the male function may be context dependent. To our knowledge, this is the first evidence of gender differences in chemical defenses of a gynodioecious species in the field.