A previous study examined the sexual expression of cultured gametophytes of the Hawaiian endemics Sadleria cyatheoides and S. pallida grown on mineral-enriched agar. In the present work, we conducted field studies on the sexual expression of natural populations of Sadleria spp. gametophytes. Our primary goal was to compare field-collected data to the laboratory-based data of the earlier study to assess if the laboratory data were an accurate reflection of what is occurring in nature. Our results suggested that for generally inferring mating systems operating in nature, agar-based laboratory studies of gametophytes lead to the same conclusions as would observations of field-collected gametophytes. For detailed studies of gametophyte sexuality and development, however, an agar-based medium produces significantly different results than what is found among natural populations, although this may be true for any lab-based study regardless of growth medium. Thus, we suggest caution in the use of agar as a growth medium, and the use of laboratory conditions in general, for studies of fern gametophyte sexual development.