Rhamnus persicifolia Moris is an endemic small tree belonging to the Rhamnus cathartica group, growing along mountainous streams of Central-Eastern Sardinia (Italy). ISSR markers were used to detect the genetic diversity within and among six populations representative of the species distribution range. In spite of the limited distribution of this endemic taxon, fairly high levels of genetic diversity were detected. Percentage of polymorphic bands (PPB), gene diversity (HS and HT) and Shannon information measure (Sh) were calculated both at population (PPB ¼ 30.70%, HS ¼ 0.1105, Sh ¼ 0.1646) and at species level (PPB ¼ 68.42%, HT ¼ 0.2066, Sh ¼ 0.3139). The existence of a spatial distribution of genetic diversity in R. persicifolia was revealed by a low gene flow, a fairly high level of genetic differentiation (GST ¼ 0.4583) among populations and a positive correlation between genetic and geographic distances (Mantel test, r ¼ 0.71, p ¼ 0.016). The spatial genetic structure was also confirmed with BAPS analysis. Our results show that a certain level of isolation by distance and sex-ratio bias may explain the distribution of genetic diversity among populations. Conservation measures are suggested on the basis of the genetic diversity detected, by implementing an integrated in situ and ex situ conservation program for each population, in order to ensure effective protection for this endemic species.