A detailed cytogeographic and morphometric study of the Asplenium trichomanes group in the Czech Republic is presented. We detected diploid (2n = 72), tetraploid (2n = 144) and hybrid triploid plants (2n = 108). Based on the morphometric study, four intraspecific taxa are recognized. These taxa correspond to the four subspecies of A. trichomanes (A. t.subsp. trichomanes, <I.A. t. subsp. quadrivalens, A. t. subsp. pachyrachis and A. t. subsp. hastatum) distinguished in the floras of western, southern and northern Europe. Triploid plants were determined as A. t. nothosubsp. lusaticum (A. t. subsp. trichomanes × A. t. subsp. quadrivalens). The individual morphological characters used for determining subspecies are evaluated and a determination key presented.