Pleurothallidinae is the largest Orchidaceae subtribe of the Neotropics, with most of its diversity composed of epiphytes from wet forests. Recent classifications still do not have sufficient micromorphological support, and genera delimitation is unclear. Here, we provide a structural analysis of the root anatomy in 55 species and 10 genera of Brazilian Pleurothallidinae and phylogenetic analysis based on ITS and MatK sequences available in databases. All roots are velamentous, with 2-6 layers of epidermal tissue. In the cortex, chloroplasts, amyloplasts, fungal pelotons, idioblasts with raphides, and tracheoid idioblasts are frequent. Exodermis and endodermis cells may be thickened in different patterns, with thin-walled passage cells. We identified four different clades: clade A (Octomeria species, ancestor with a two-layered velamen, without tilosomes, U-thickened exodermis, and sclerified pith), clade B (Echinosepala and Myoxanthus, ancestor with three or more layers in the velamen, without tilosomes, more than five layers of cortical parenchyma, pith with amyloplast), Clade C (Anathallis, Masdevallia, Pabstiella, Specklinia, Stelis, and Zootrophion; ancestor with two-layered velamen, with tilosomes, thin-walled exodermis, sclerified pith), and clade D (Acianthera, ancestor with two-layered velamen, without tilosomes, O-thickened exodermis, up to five layers of cortical parenchyma, sclerified pith). The traits cited for these clades are not necessarily apomorphies and may have been lost in some of the subclades. However, it indicates the ancestral characteristics of the group and can contribute to its systematics. The relationships in Pleurothallidinae and its large number of species require multidisciplinary attention to the group to better understand the group's evolution, including more structural analyses like this study.