Habitat fragmentation is one of the main threats to semi-natural grasslands all over Europe, causing reductions in both numbers of populations and numbers of individuals withinpopulations. Small isolated populations are particularly vulnerable to fluctuations that reducespopulation size and could lead to loss of genetic variation due to inbreeding depression, orfixation of harmful alleles due to genetic drift. The aim of this study was to investigate if thecritically endangered eyebright Euphrasia stricta var. suecica experiences pollen limitation,inbreeding depression and heterosis or outbreeding depression. Low pollen limitation waspredicted as well as low inbreeding depression due to high selfing rate. Outbreedingdepression or heterosis was expected based on previous studies indicating strong geneticdifferentiation among populations. This study was conducted in two traditionally managedmeadows in the central part of Gotland, Sweden, using supplemental hand-pollinations andcontrolled crosses. Individuals were collected when the flowering period was over and seedswere counted and weighed. There were no signs of either pollen limitation, inbreedingdepression, heterosis or outbreeding depression in any of the two wooded hay meadows. Thedegree of autonomous selfing was high, indicating that E. stricta var. suecica is not dependenton pollinators for seed production. A significant difference between the meadows was foundin proportion of viable seeds. As there were no signs of local adaptation, individuals could betranslocated between the two studied populations or seed sowings could be used to increasepopulation size. However, before such conservation efforts are undertaken, it is important toconduct further studies that investigate several populations including more life-stages.