The Agasthyamala Biosphere Reserve (ABR), situated at the southern extremity of the Western Ghats, represents one of the most important centres of tropical biodiversity and endemism in peninsular India. Despite more than a century of botanical exploration, information on its angiosperm diversity remains scattered across numerous floras, checklists, unpublished reports and continuously changing taxonomic databases, leading to inconsistencies in nomenclature and distributional records. The present study addresses this gap by providing a critically compiled and taxonomically updated checklist of the flowering plants of the ABR based on an extensive evaluation of original protologues, classical floras, recent literature and global taxonomic repositories following the Angiosperm Phylogeny Group IV classification system. The study documented 3261 species, 26 subspecies and 28 varieties belonging to 1139 genera and 190 families, making ABR one of the richest floristic regions in peninsular India. Fabaceae emerged as the most species-rich family with 349 taxa, followed by Rubiaceae (194 taxa), Poaceae (193 taxa) and Orchidaceae (168 taxa). A remarkably high level of endemism was recorded, with 1018 taxa (31.2 %) endemic to India, including 245 steno-endemic taxa confined to highly specialised habitats and narrow altitudinal zones. The flora also revealed strong biogeographic affinity with Sri Lanka, with 347 taxa shared between the two regions, highlighting the significance of the southern Western Ghats–Sri Lanka floristic province. The coexistence of ancient lineages and recently diversified endemic taxa indicates that the Agasthyamala landscape functions both as an evolutionary refugium and as an active centre of speciation. These findings reaffirm the exceptional ecological and evolutionary significance of the ABR and emphasise the vulnerability of its habitat-specific flora to ongoing anthropogenic pressures and climate change. The study provides an authoritative baseline for biodiversity assessment and demonstrates that effective conservation of the ABR requires targeted habitat protection, long-term monitoring and region-specific management strategies to safeguard one of the world's most important centres of tropical plant diversity.