Flow cytometry measurements confirmed the occurrence of Polypodium ×mantoniae (P. interjectum × P. vulgare) at three localities in the eastern part of the Czech Republic (Blansko and Rudice N of Brno and Javoříčko WNW of Olomouc). Nuclear DNA contents (± Sx) were determined for P. vulgare (2C = 29.00 ± 0.32 pg), P. ×mantoniae (2C = 37.18 ± 0.38 pg) and P. interjectum (2C = 45.24 ± 0.31 pg) using a PAS Partec GmbH flow cytometer (PI staining / standard Vicia faba, 2C = 26.9 pg). The relative DNA content ratio was measured in all pairs of taxa (± Sx range), i.e. P. ×mantoniae : P. vulgare = 1.340 ± 0.008; P. interjectum : P. vulgare = 1.681 ± 0.003; P. interjectum : P. ×mantoniae = 1.255 ± 0.008. Six new localities for Polypodium interjectum were found in the region of Moravský Kras (= Moravian Karst, N of Brno). From the PI/DAPI index it can be inferred that the AT/GC ratio (or heterochromatin occurrence) is 1.05× bigger in P. ×mantoniae than in P. vulgare and 1.08× bigger in P. interjectum than in P. vulgare. Anatomical data (number of thick- walled cells in the anulus, spore length and stomata length) of selected specimens and live samples from the Czech Republic were in good agreement with the range of variation of these features published by earlier authors from other European countries. A brief historical survey of the knowledge of P. interjectum in the Czech Republic is included.