As has been shown for many kinds of organisms, barriers to interspecific hybridization may differ in strength between reciprocal crosses, resulting in a bias in the probability that one or the other species may be the maternal or paternal parent of hybrids. The fern Dryopteris Xtriploidea, the "backcross" hybrid between the diploid D. intermedia and the tetraploid D. carthusiana, occurs in large numbers in nature, providing an opportunity to investigate whether such a bias exists. Differences in the chloroplast genome distinguishing the two parental species were discovered in the sequence of the trnL region following amplification by polymerase chain reaction (PCR), including a Mse I restriction site. This allowed rapid identification of the donor of the chloroplast genome, and therefore the maternal parent of each hybrid, assuming chloroplast DNA to be maternally inherited in Dryopteris. Analysis was carried out on 127 hybrids, shown to be of independent origin using allozymes, occurring at three localities in Virginia and West Virginia. When samples from all localities were pooled, 91 possessed the D. carthusiana trnL genotype and 36 possessed the D. intermedia genotype, a ratio that is significantly different (P < 0.001) from the null hypothesis of no gender bias. The strength of the bias differed significantly among the three sites, however, with bias at the West Virginia site much stronger (5.6:1 carthusiana:intermedia; P < 0.001) than at either Virginia site (1.55:1 and 1.43:1 carthusiana:intermedia, respectively; P > 0.05 in both cases). The cause of the strong bias in the West Virginia sample is unknown, as is the cause of the population differences. Causes of bias could include differences between the parental species related to their ploidy difference, including sizes of gametes and/or gametangia, sperm motility, breeding system (D. intermedia is outcrossing while D. carthusiana is selfing), or the nature and strength of interspecific isolating mechanisms.