Diphasiastrum comprises 20–30 species. In addition to a number of species with a circumboreal distribution, several island endemics and putative diploid hybrid species contribute to the diversity of the group. To assess the integrity and relationships of the recognized species, a global phylogeny of Diphasiastrum is constructed using five chloroplast regions comprising ca. 9,000 bp. Six monophyletic groups are established. Accessions identified as hybrid species cluster in all but one case together with one of its putative parents. Allelic information combined with sequence information obtained from two chloroplast microsatellite loci is found diagnostic for the three putative parental taxa in the Central Europe hybrid complexes. Haplotype screening is performed on six Central European populations, from where one or more putative diploid hybrid species have been reported to grow in sympatry with their parent species. The most common parental haplotypes are identified in all populations. Additional intraspecific variation, restricted to single populations, is identified in all sympatric populations at very low frequencies. Taking the low degree of sequence and microsatellite variation into consideration, the acknowledged morphological diversity in Central Europe is probably best explained by phenotypic plasticity, ancestral polymorphisms or relatively recent events of reticulate evolution.