Spathoglottis plicata is an endangered terrestrial orchid species that have experienced severe threats to its habitat as wild forest sites come under pressure from industrialisation and natural disasters. This orchid species chosen to evaluate their levels of genetic diversity and population genetic structure, which 25-30 accession collected in the different location with different geographical, altitude and habitat. Genomic DNA was extracted from six natural populations (n=172) in Peninsular Malaysia using eleven AFLP markers of EcoRI+3 bases/MseI+3 base primer combinations. Based on 279 polymorphic bands, a significant degree of genetic population differentiation was found, with a 78.5% variation within populations as measured by AMOVA, indicating a potential restricted gene flow. Two distinct clades generated from a UPGMA dendrogram were further investigated through a Bayesian analysis using STRUCTURE software, producing an estimated population structure at optimal value K=4. These results point to the presence of four genetic structures in the Spathoglottis plicata population. The Pahang and Terengganu population revealed a higher than average genetic variation (60.25%), indicating that there may be a robust structural division between the population samples and a possible hybridisation between the Northern (Kedah), Southern (Negeri Sembilan and Johor) and Central (Selangor) region populations. In sum, these results suggest that geographical distance is the primary factor contributing to differences among populations and the need for conservation measures to protect the Spathoglottis plicata species.