The Giant Freshwater Prawn (GFP), Macrobrachium rosenbergii, is a cornerstone of global freshwater aquaculture, especially in Southeast Asia, due to its increasing demand for high-quality seed stock and market value, yet the genetic structure of its natural populations in Malaysia remains poorly understood. This study evaluated the population structure and genetic diversity in the wild GFP across Peninsular Malaysia to identify superior strains for sustainable breeding programmes. This study analysed a 572 base pair segment of the mitochondrial cytochrome c oxidase 1 (COX1) gene from 149 GFP individuals collected from six river systems across Peninsular Malaysia. Twelve distinct haplotypes were identified, with phylogenetic analysis showing polyphyletic relationships among populations. The Kelantan river population exhibited the highest level of genetic diversity (π = 0.0024) and haplotype diversity (h = 0.5885), whereas the Perak river population showed no polymorphism. These results suggest a historical population bottleneck that drastically reduces the effective population size, in turn leading to loss of rare alleles and thus the increase in genetic homogeneity. As indicated by negative Tajima’s D and Fu’s FS values, significant deviations from neutrality, together with unimodal mismatch distributions, suggest a historical bottleneck followed by rapid population growth and the subsequent accumulation of rare mutations. These findings provide a critical genetic baseline for the management of wild stocks and the selection of high-diversity founder populations for the GFP aquaculture industry in Malaysia.