Lotus (Nelumbo Adans.) is an important ornamental plant and a commercial crop that is distributed widely in Asia and northern Australia. Wild lotus is suffering from habitat loss because of global environmental change and intensive human activities. Conservation of wild lotus requires protection of genetic resources. To assess the genetic diversity of wild lotus worldwide, 83 individuals from populations in America, Thailand, and China were analyzed with simple sequence repeat (SSR) markers. A total of 145 alleles were identified with 36 SSR markers in all accessions. Analysis of molecular variance (AMOVA) showed that the higher genetic diversity was observed among populations (88%) than within populations (12%). A high degree of genetic differentiation was identified among the 11 populations (average FST = 0.42). Low genetic diversity was revealed within each population, which possibly resulted from predominant asexual reproduction of this species. Mantel’s test demonstrated that genetic differentiation among populations was congruent with geographical distance (r = 0.89, P < 0.001), which indicated that isolation-by-distance was responsible for the observed genetic structure of the populations of Nelumbo analyzed. Cluster, principal coordinate and STRUCTURE analyses revealed that the American populations (Nelumbo lutea Pers.) were genetically distinct from those from Thailand and China (Nelumbo nucifera Gaertn.), and confirmed the genetic distinction between N. nucifera and N. lutea. Moreover, the Thai genotypes were separated from the Chinese genotypes, which represented two ecotypes of N. nucifera. Given the high level of genetic differentiation among populations, every population should be considered for conservation. In situ preservation is the preferred strategy to conserve the natural genetic diversity and evolutionary processes of Nelumbo, but should be supplemented by ex situ conservation.