Weedy rice has been becoming a notorious weed in the paddy field of China in recent decades due to its increasing damage to rice yield and rice quality. In this study, a microsatellite technique with 21 pairs of SSR markers was utilized to estimate the genetic structure of two biotypes of weedy rice with Japonica and Indica rice characteristics, collected from Liaoning and Guangdong provinces, respectively. The genetic diversity of the weedy rice in the two provinces was relatively low (Liaoning h = 0.086; Guangdong h = 0.160), and distinctly large genetic differences existed between these two provinces (Gcs = 0.623). The genetic diversity was found primarily within populations, and genetic differentiation was relatively low within the same province. Both cluster analysis (UPGMA) and principle component analysis (PCA) showed that weedy rice had a closer relationship with the cultivated rice collected from the sample field than with other cultivated rice and common wild rice varieties in China. Thus, the results of this study on samples from the Liaoning and Guangdong provinces in China support the de-domestication hypothesis that weedy rice most probably originated from local cultivated rice.