In this study, mating system, genetic diversity, and genetic structure of the endangered endemic aquatic lycophyte Isoëtes yunguiensis in China was investigated using AFLP markers. The estimates of outcrossing rates (t_m = 1.200) indicate that diploid I. yunguiensis is a predominantly outcrossing species. Six selected AFLP primer pairs used in the study amplified 195 reproducible bands, 160 of which were polymorphic (PPB = 82.05%), indicating significant genetic diversity at the species level. AMOVA revealed that 40.12% of the genetic variation was attributable to differences between populations and the rest (59.88%) to variability within populations. High outcrossing rates and the historical accumulation of genetic variation may be responsible for the high levels of genetic diversity observed in the I. yunguiensis population. To maintain the current level of genetic diversity for this species, we recommend increasing in situ conservation sites.