Swat Valley, located in Koh Hindu Kush-Himalayan region of Pakistan, is rich in floral diversity. However, climate change is impacting and putting plant diversity at risk, leading to elimination and even extinction of many vulnerable plant species. Effective conservation and monitoring therefore require reliable tools for species identification. Ferns are a key component of the valley’s temperate vegetation; however, despite extensive morphological studies, their molecular characterization and phylogenetic relationships remain poorly investigated. Ferns constitute an important component of the valley’s temperate vegetation, yet despite extensive morphological work, their phylogenetic relationships and molecular characterization remain largely unexplored and sketchy. This study was undertaken to DNA-barcode two Adiantum species and to assess their genetic variability and phylogenetic relationships. Two loci, rbcLa and psbA-trnH were amplified through polymerase chain reaction (PCR) and sequenced using Sanger method. Morphological diagnosis, combined with BLASTn homology searches, K2P genetic distance analyses, and phylogenetic reconstruction, consistently identified the fern species as Adiantum capillus-veneris L. and A. venustum D. Don. The results demonstrated the complementary effectiveness of rbcLa and psbA-trnH for accurate identification and monitoring of regional Adiantum species and revealed two likely misannotated sequences in GenBank (MF694655.1; MF694942.1) and one in BOLD systems (DBMPP-195-14). The study also suggested further morphological and multilocus investigation of A. capillus-veneris L. and A. ogasawarense Tagawa, as two markers used in this study appear to be highly conserved between them. The validated sequences have been deposited in NCBI database and accessions assigned to support future taxonomic, phylogenetic, and conservation studies of the genus.