Traditionally, new plant species identification relied primarily on morphological characteristics. However, molecular methods have now been demonstrated to provide more reliable identification. Cyrtomium adenotrichum, a fern species discovered in the Nandan limestone area of Guangxi, China, was initially described as a new species based on its distinct taxonomic morphology. To investigate the phylogenetic position of this newly described species, we conducted molecular phylogenetic analyses using the rbcL and psbA-trnH gene regions. Our study aimed to evaluate the effectiveness of these DNA barcodes in distinguishing C. adenotrichum from closely related species. Maximum likelihood phylogenetic reconstruction revealed that both barcode regions clearly differentiated C. adenotrichum from its congeners, with significant topological divergence observed in the resulting trees. These findings demonstrate that rbcL and psbA-trnH serve as highly effective molecular markers for the accurate identification of C. adenotrichum.