Premise of the study:Polymorphic microRNA (miRNA)–based microsatellite markers were developed to investigate the genetic diversity and population structure of Nelumbo nucifera (Nelumbonaceae).Methods and Results:A total of 485 miRNA‐based microsatellites were found from the genomic DNA sequences of N. nucifera. After several rounds of screening, 21 primer pairs flanking di‐, tri‐, or pentanucleotide repeats were identified that revealed high levels of genetic diversity in four populations with two to five alleles per locus. The observed and expected heterozygosity per locus ranged from 0.000 to 1.000 and from 0.000 to 0.803, respectively.Conclusions:The polymorphic microsatellite markers will be useful for studying the genetic diversity and population structure of N. nucifera.