Wild rice Zizania latifolia is a perennial emergent aquatic plant widely distributed across China. Wild populations of Z. latifolia are important to aquatic ecosystems and are valuable genetic resources for breeding. However, they have been faced with significant habitat losses in recent decades. For 10 nuclear microsatellites, high levels of genetic diversity (HE = 0.572–0.636) were found across seven surveyed populations from central China. The main factors responsible for that were its long life history and predominant outcrossing reproductive system. Low genetic differentiation among populations was found based on Wright's FST = 0.098. Similarly, AMOVA analysis showed only 7.73% of the total molecular variation was attributed to inter-population differentiation. The weak population structure of Z. latifolia could be due to high gene flow mediated by water or birds (Nm = 2.30, M = 2.18). Importantly, most populations exhibited mutation-drift disequilibrium, suggesting a recent population decline. Based on the results, wild populations of Z. latifolia are expected to lose genetic diversity and increase genetic structure in future generations. Therefore, conservation management is urgently needed to maintain the genetic resources of Z. latifolia.