Abstract: To determine the genetic structure of natural Myriophyllum spicatum populations in mainland China and the possible mechanism that led to the widely distributed pattern of the species, eight populations were examined for preliminary genetic analysis based on three chloroplast DNA segments (trnK-matK, trnL-trnF, rpl32-trnL). The AMOVA analysis results showed that the genetic diversity among the eight populations was 84.97% and within the populations was 15.03%. Furthermore, the population genetic differentiation coefficient (Fst) was 0.85, indicating high genetic diversity in M. spicatum (Hd=0.83) among populations. The founder effect may have led to the initial genetic differences and segregation barriers (Nm=0.09) may have further resulted in genetic differentiation among populations. From the constructed phylogenetic tree and haplotype network based on 17 haplotypes, haplotypes H5 and H6 had the highest frequency and widest distribution, indicating they were possibly the oldest ancestral haplotypes. The Mantel test showed no significant correlation between genetic and geographic distances among populations, and the mismatch distribution test showed that historic M. spicatum populations likely experienced expansion events. Tajima's, Fu & Li's D^* and F^* tests showed that this species did not have an obvious phylogeographic pattern, which may have resulted from long distance dispersal of seeds.