<p>Although knowledge of arbuscular mycorrhizal fungi (AMF) in leaf litter remains limited, reports of their presence in this substrate have become increasingly frequent. However, the factors driving litter colonization and the identity of AMF species occurring in such conditions are still poorly understood. It has been suggested that these fungi may preferentially colonize litter during the early stages of decomposition. However, to date, no study has identified AMF species in this substrate, leaving their diversity, ecological roles, and contribution to decomposition largely unresolved. In light of this, leaf litter samples were collected from three Brazilian Atlantic Forest sites and analyzed using morphological and molecular approaches. According to the analysis, the bag exposure duration, litter quality (determined by a set of chemical: concentrations of carbon, nitrogen, phosphorus and physical traits of leaf (specific leaf area). Leaves that have higher stoichiometric ratios such as C:N and C:P and lower SLA are determined as a lower quality litter, while those having high concentrations of nitrogen and phosphorus and high SLA are determined as a high quality litter), and edaphic factors drove AMF colonization in leaf litter. <i>Diversispora varaderana</i>, <i>Funneliformis caledonius</i>, <i>F. geosporus</i>, <i>Glomus spinuliferum</i>, and <i>Rhizoglomus irregulare</i> were reported for the first time in association with leaf litter. Furthermore, <i>D. varaderana</i> and <i>F. caledonius</i> represented new records for Brazil and the Brazilian Atlantic Forest, respectively. These findings highlight leaf litter as a promising habitat, complementing previous studies on soils for AMF diversity and their ecological role in decomposition, along with its biotechnological applications.</p>