American lotus (Nelumbo lutea Willd.) is a popular plant with ornamental, medicinal, and culinary value. Studies related to the genetic diversity and population structure of N. lutea are limited. We sampled 326 individuals from 19 populations of wild N. lutea across its distribution range in the USA and genotyped them using 12 microsatellite markers. A total of 274 alleles were amplified by 12 microsatellites with a mean of 22.83. We observed a low level of genetic diversity for American lotus (Ae=2.40 and HE=0.459) in the USA. The analysis of molecular variance (AMOVA) exhibited closer variation among populations (42.00%) than within populations (58.00%) of N. lutea, with strong genetic differentiation observed (FST=0.419). There was no significant correlation between genetic and geographic distances. The Bayesian analysis and neighbor-joining dendrogram displayed strong population structure. A very low level of historical gene flow (NM=0.073) was observed among the populations. Fourteen of the 19 populations showed a demographic bottleneck, indicating the vulnerability of the American lotus populations. We thus proposed priority population sites for the conservation of N. lutea via both ex-situ and in-situ techniques.