Myricaria laxiflora was once considered to be restricted to the Three Gorges Reservoir Area (TGRA), China, and was thought to be the only species to become extinct due to the construction of the Three Gorges Dam (TGD). Recently, three natural populations of this species were found along the Yangtze River valley downstream of the TGD. We studied the genetic diversity and genetic differentiation of three remnant populations and nine extirpated populations, the material of which were collected from natural populations before the impoundment of the Three Gorges Reservoir, using inter-simple sequence repeat (ISSR). High levels of genetic diversity were revealed within populations, both at the species [percentage of polymorphic bands (PPB) = 95.9%] and population level (PPB varied from 58.5% to 80.3%). For the three remnant populations, the Yidu (MYD) and Yichang (MYC) populations showed comparatively higher genetic diversity among the 12 populations, whereas the Zhijiang (MZJ) population showed lower genetic variation. 97.96% of total genetic variation of the species was contained in the three remnant populations. The MZJ population appeared to be distinct from the other 11 populations, whereas the MYD and MYC populations had a close genetic relationship with the nine extirpated populations. A moderate level of genetic differentiation was detected among populations by different estimation approaches. The high genetic diversity of the three natural remnant populations indicates that in situ conservation of this species may enable it to successfully maintain its evolutionary potential, especially in the present case that the habitat of transplanted populations are not suitable for individuals’ survival.