Abstract Distribution of genetic diversity among and within plant populations may depend on the mating system and the mechanisms underlying the efficiency of pollen and seed dispersal. In self-incompatible species, negative frequency-dependent selection acting on the self-incompatibility locus is expected to decrease intensity of spatial genetic structure (SGS) and to reduce population differentiation. We investigated two populations (peripheral and more central) of wild service tree (Sorbus torminalis (L.) Crantz), a self-incompatible, scattered tree species to test the differences in population differentiation and spatial genetic structure assessed at the self-incompatibility locus and neutral nuclear microsatellites. Although, both populations exhibited similar levels of genetic diversity regardless of the marker type, significant differentiation was noticed. Differences between FST and RST suggested that in the case of microsatellites both mutations and drift were responsible for the observed differentiation level, but in the case of the S-RNase locus drift played a major role. Microsatellites indicated a similar and significant level of spatial genetic structure in both populations; however, at the S-RNase locus significant spatial genetic structure was found only in the fragmented population located at the north-eastern species range limits. Differences in SGS between the populations detected at the self-incompatibility locus were attributed mainly to the differences in fragmentation and population history.