Estrogens play a significant role in bone physiology. Their action is mainly exerted through their receptors. Estrogen receptor alpha (ERα) plays a major role in bone homeostasis and there is evidence suggesting that estrogen receptor beta (ERβ) has also an effect on BMD. We investigated the possible effect of two ERβ gene polymorphisms on spinal bone mineral density (BMD) and metabolic bone markers in Greek women. Spine BMD as well as biochemical bone markers were measured in 147 healthy peri- and post-menopausal women [mean age (S.D.) 54 (7.9) years]. Genotyping was performed for two restriction fragment length polymorphisms (RFLPs) of ERβ gene, RsaI in exon 5 and AluI in exon 8. For each polymorphism studied the cohort was divided into two groups: the “wild-type” group (RR and AA, respectively) and the “carrier” group including subjects with at least one allele with the restriction site (Rr&rr and Aa&aa, respectively). The distribution of RsaI genotypes was RR: 91.2% ( n = 134), Rr: 8.2% ( n = 12), and rr: 0.6% ( n = 1) and of AluI genotypes AA: 36.7% ( n = 54), Aa: 57.2% ( n = 84), and aa: 6.1% ( n = 9). No linkage disequilibrium was found between the two polymorphic sites studied. Spine BMD did not differ significantly in the two groups of either polymorphism, after adjusting for age, weight, height, and years since menopause [mean BMD (S.D.) for RR 0.841 (0.17) g/cm 2 versus Rr&rr 0.798 (0.13) g/cm 2 , p = 0.25, and mean BMD (S.D.) for AA 0.828 (0.16) g/cm 2 versus Aa&aa 0.848 (0.17) g/cm 2 , p = 0.32]. No significant differences were noted in metabolic bone markers except for a marginal difference of RR versus Rr/rr in urinary hydroxyproline/creatinine ratio [median (IQR) 3.88 (6.04) μmol/mmol in RR versus 8.2 (4.32) μmol/mmol in Rr/rr, p = 0.05]. Furthermore, no interaction between the two polymorphisms on BMD was found. In conclusion, in a Greek female post-menopausal population, the two ERβ gene polymorphisms were not associated with BMD, or metabolic bone markers.