Mating system has important implications for the genetic structure and diversity of populations, especially threatened and endangered species. In this study, mating system of the endangered aquatic fern Ceratopteris pteridoides in China was investigated using AFLP markers and selfing test. The results of AFLP analyses showed that the estimate of multilocus Soutcrossing rate (t(m)) was high at species level (t(m) = 0.999), indicating that C. pteridoides is a predominant outcrossing species. The small differences between t(m) and t(s) (0.136 +/- 0.003) at species level showed that there is a low tendency for mating between relatives. The selfing test showed that isolated gametophytes formed the rate of normal sporophytes in three populations (BLH, ZDH, WCH) ranging from 72.9% to 77.8% with a mean value of 75.2%, indicating that C. pteridoides has a high level of intragametophytic selfing potential. Our investigation found that some of the studied populations of C. pteridoides have a prolific capacity for vegetative reproduction (clonal growth). The results revealed that the diploid homosporous pteridophyte C. pteridoides predominantly favors gametophytic crossing with a certain level of inbreeding along with clonal growth. We suggest that mating system of C. pteridoides may be adjusted to promote selfing rate in response to factors including the characteristics of hermaphrodite, and colonization events and small extant populations. As a result of the high outcrossing rate of C. pteridoides, we recommend that ex situ plantings would be most suitable for enhancing possible intermingling of the different populations thus minimizing inbreeding.