Morphological and hemodynamic variations of the circle of Willis (CW) may have an important impact on cerebrovascular events. However, the environmental and genetic influence remains unclear. For this reason we studied the variations and hemodynamic parameters of the CW in twins using transcranial color-coded sonography (TCCS). Sixty-four twins, 19 monozygotic (MZ) and 13 dizygotic (DZ) pairs from the Italian Twin Registry (average age 45.0 ± 13.7 years) underwent TCCS and risk factor assessment. We examined CW morphology and recorded peak systolic velocity (PSV), end-diastolic velocity (EDV) and pulsatility index (PI). Raw heritability was determined for hemodynamic parameters, whereas concordance and discordance rates were calculated for CW morphological variants. A normal CW anatomy was observed in the majority of MZ and DZ twins (76.5% and 92.3%, respectively). The most frequent variant was a missing anterior cerebral artery (ACA). There was no significant difference in the prevalence of most CW variants depending on the zigosity. Concordance rates were low regarding the presence of variant CW anatomy both in MZ and DZ groups (0.14 and 0.00, respectively). Women had a significantly higher PI in vertebral arteries (VA) and in the right ACA (p = 0.01, p = 0.02 and p < 0.01, respectively). An inverse correlation was observed between hemodynamic parameters and age. Morphological variants of the CW do not seem to be heritable; they are most likely determined by environmental factors. In contrast, hemodynamic parameters of the CW are moderately heritable and this might have implications in the management and prevention of cerebrovascular diseases.