Several cytotypes (polyploids and aneuploids) have been reported in Lepisorus thunbergianus. The relationships between these cytotypes within the species remain poorly understood. We studied populations in an area where various cytotypes of L. thunbergianus as well as two diploid species, L. angustus and L. onoei, candidate parental species that may be involved in allopolyploid origins of L. thunbergianus polyploids, occur. We determined the ploidy levels of sampled materials by direct chromosome counting and flow cytometry. We elucidated the origins of L. thunbergianus polyploids by analyzing allozyme polymorphisms, and in addition, we examined the occurrence of segmental allopolyploidy by comparing allelic variation between polyploids and their parental diploids. Six cytotypes, i.e. one diploid (2n = 50), two triploid (2n = 75 and 76) and three tetraploid (2n = 100, 101, and 102) cytotypes, were observed in L. thunbergianus, and the two diploid species, L. angustus (2n =52) and L. onoei (2n =50) were confirmed to include a single cytotype each. Allozyme analyses indicated that the tetraploid (2n =100) and hypertetraploid (2n = 102) of L. thunbergianus originated by allopolyploidy between diploid L. thunbergianus (2n = 50) and diploid L. angustus (2n = 52), since the polyploids shared alleles with these two diploids that were unique to each diploid. The allozyme patterns excluded the possibility that L. thunbergianus polyploids originated from L. onoei. The unbalanced heterozygosity and homozygosity found in the tetraploid and the hypertetraploid of L. thunbergianus indicated their segmental allopolyploidy.