Premise The gametophytes of different fern species collected in the field can be difficult to distinguish because of their morphological similarities. Nonetheless, emerging molecular ecology techniques are starting to be used to tackle such limitations. Here, using case studies and a detailed protocol, we demonstrate a convenient methodology, tissue‐direct PCR (TD‐PCR), that foregoes a traditional DNA extraction and facilitates the identification of fern gametophytes, as well as enabling the elucidation of their natural distribution. Methods Based on updated plastome information, we designed a universal primer set targeting the trnL‐L‐F region, which is effective across extant ferns. We used this primer set to perform TD‐PCR on the case‐studied populations of Taiwanese Lomariopsis gametophytes, using the generated sequences for their identification. In the case study concerning the microhabitat preference of Vaginularia junghuhnii , we designed and used a taxon‐specific primer set. Results Compared with approaches requiring DNA extraction, the use of TD‐PCR with either universal or taxon‐specific primers could save significant time, money, labor, and research materials in the genetic identification of fern gametophytes. Discussion The use of modern genetic tools can aid in the identification of fern gametophytes. An updated TD‐PCR strategy not only facilitates the DNA‐based identification of gametophytes, but also promotes new avenues of research for investigating these plants in the field.

Premise Efficient protocols for extracting high‐molecular‐weight (HMW) DNA from ferns facilitate the long‐read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)‐based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time. Methods and Results We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33 species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8). Conclusions This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.