Abstract Allozyme markers were used to characterize spatial genetic structure for the weedy fern Pteris multifida within three populations growing on the man made stone walls. Even visually distinct genetic substructures were observed in all the examined populations. Spatial autocorrelation analysis using Moran's /indicated that the patch size of P. multifida was as small as 4–9 m. Estimation of outcrossing rate is known to be severely biased downwards if the presence of spatial genetic structure is ignored. Mating system of this fern species was reported as mixed (mean selfing rate: 0.435). However, fixation indexes calculated in small patches less than 3–10 m wide were not significantly different from Hardy-Weinberg equilibrium and thus P. multifida's mating system was considered to be outcrossing.