Comprehending genetic variation and population structure is paramount for devising optimal management strategies and conservation efforts. In this study, we employed amplified fragment length polymorphism (AFLP) analysis to assess the distribution of genetic variation within and among populations of the rare macrophyte <i>Potamogeton rutilus</i> Wolfg. This species has experienced habitat alterations leading to its disappearance from numerous previously known European locations. Our analysis encompassed eight populations collected from Poland, Lithuania, and Estonia, situated within the central region of <i>P. rutilus</i> distribution. The results of our study indicate that the analyzed populations are characterized by relatively low levels of genetic diversity (the mean of Nei’s gene diversity was 0.031±0.003 and the Shannon’s information index 0.049± 0.004), and intermediate gene flow (Nm = 0.289). Analysis of molecular variance (AMOVA) revealed higher levels of variation among populations (63%) than within populations (37%); PhiPT = 0.634, <i>p</i> < 0.001, which might be attributed to the founder effect, frequent vegetative propagation, and limited seedling recruitment. The results of STRUCTURE, PCoA and NeighbourNet analyses suggest that the genetic structure of the studied populations falls into three main clusters not fully corresponding to geographical regions. The Mantel test did not show a significant correlation between genetic distance and geographical distance among populations. The most geographically isolated population sampled in Poland (ROT) is genetically close to the Estonian population VAL and VIT, although it is located far closer to the stations in Lithuania. Implications of acquired data for the genetic management of the species are discussed.