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Cycas multipinnata C.J. Chen & S.Y. Yang is a cycad endemic to the Red River drainage region that occurs under evergreen forest on steep limestone slopes in Southwest China and northern Vietnam. It is listed as endangered due to habitat loss and over-collecting for the ornamental plant trade, and only several populations remain. In this study, we assess the genetic variation, population structure, and phylogeography of C. multipinnata populations to help develop strategies for the conservation of the species. 60 individuals from six populations were used for chloroplast DNA (cpDNA) sequencing and 100 individuals from five populations were genotyped using 17 nuclear microsatellites. High genetic differentiation among populations was detected, suggesting that pollen or seed dispersal was restricted within populations. Two main genetic clusters were observed in both the cpDNA and microsatellite loci, corresponding to Yunnan China and northern Vietnam. These clusters indicated low levels of gene flow between the regions since their divergence in the late Pleistocene, which was inferred from both Bayesian and coalescent analysis. In addition, the result of a Bayesian skyline plot based on cpDNA portrayed a long history of constant population size followed by a decline in the last 50,000 years of C. multipinnata that was perhaps affected by the Quaternary glaciations, a finding that was also supported by the Garza-Williamson index calculated from the microsatellite data. The genetic consequences produced by climatic oscillations and anthropogenic disturbances are considered key pressures on C. multipinnata. To establish a conservation management plan, each population of C. multipinnata should be recognized as a Management Unit (MU). In situ and ex situ actions, such as controlling overexploitation and creating a germplasm bank with high genetic diversity, should be urgently implemented to preserve this species.