The mycorrhizas of 12 species of Polygala (Polygalaceae), including herbs, subshrubs and one shrub, collected from Germany, Mallorca (Spain) and Malta, were investigated by morpho-anatomical and molecular methods. Aseptate hyphae, arbuscules and vesicles indicate an arbuscular mycorrhiza in all species examined. Hyphal spread in Polygala is predominantly, but not exclusively, intracellular and comprises three characteristic stages of colonization: (i) intracellular, linear hyphal growth in a cascading manner after penetration towards the penultimate parenchyma layer (layer 2), (ii) initially linear hyphal growth in the cells of layer 2 from where hyphal branches repeatedly penetrate the anatomically distinct innermost parenchyma layer (layer 1), forming arbuscule-like structures therein which are subject to degeneration, (iii) more branches from the linear hyphae in layer 2 develop, but coil and make contact to the layer outside layer 2 (layer 3) in which arbuscule-like structures similar to those in layer 1 form and degenerate. This general colonization pattern differs in details between the species, and critical comparisons, in particular between the woody P. myrtifolia, the herbaceous Polygala spp. and the mycoheterotrophic Epirixanthes spp. (Polygalaceae) suggest an evolutionary shift of mycorrhizal features within the family towards an optimization of plant benefit through the fungus. Based on the molecular marker 18S rDNA mycorrhizal fungi detected in roots of Polygala spp. are largely restricted to five clades of Glomeraceae 1 (Glomus Group A). This result rejects the hypothesis of a strict symbiotic specificity in Polygalaceae but may stimulate a discussion on functionally compatible groups of fungi.