Wolffia Horkel ex Schleid. (Lemnaceae) includes the world‘s smallest angiosperms. Morphologically, their bodies are extremely simplified, making classification difficult and long-disputed. No reports about the genetic structure of these clonal-dominant aquatic plants have been published until now. In this study, of 247 samples from 42 populations spanning representative locations in China, two chloroplast haplotypes (glo-cp and un-cp) and 66 amplified fragment length polymorphism genotypes were identified. Based on wide sampling, cpDNA haplotypes, and amplified fragment length polymorphism profiles, we found that there are two genetic lineages (glo- and un- lineages) of Wolffia species in China. Genotypic and genetic diversity of Wolffia species are high compared with other clonal plants (Simpson‘s index, D= 0.97; Nei‘s diversity, H= 0.1835). Different spatial structure patterns were detected between glo- and un- lineages. Positive autocorrelation at short distances (<400 km) and slightly negative autocorrelations at larger distances (>1500 km) were detected within the glo-lineage, but no significant spatial genetic structure was detected beyond 100 km within the un-lineage. Overall, spatial genetic analysis of W. globosa revealed significant autocorrelation within short distances, indicating that restricted gene flow might be one of the most important factors in shaping genetic structure.