Aspleniaceae is a diverse fern family with around 730 species distributed across various climates. Historically, its genera were differentiated by leaf morphology, but molecular studies revealed inconsistencies with this method, leading to a revised classification into two genera. Anatomical studies, particularly of petiole color and vascular bundles, offer insight into phylogenetic relationships. Our research examines petiole anatomy to understand its link to classification and phylogeny, and to investigate the functional structures of Aspleniaceae species, thus contributing to a deeper understanding of their diversity and evolution. The present study analyzed 49 Aspleniaceae species, previously categorized into 9 genera, collected from the field and botanic gardens. Sections were stained with safranin O-fast green and examined with various microscopes to assess anatomical structures, contributing to the understanding of Aspleniaceae phylogeny. We identified three petiole types based on vascular and cellular structure. Hymenasplenium species shared common traits such as cell wall thickness in the epidermis, while Asplenium groups exhibited variation in the epidermal and hypodermal cell wall composition affecting petiole color. This research contributes to understanding phylogenetic relationships within Aspleniaceae, providing a detailed anatomical basis for classification.