Quantitative ultrasound (QUS) is a peripheral bone densitometry technique that is rapidly gaining in popularity, and is widely used worldwide for the assessment of skeletal status. This, however, generally occurs in the absence of adequate clinical guidelines. As accurate interpretation of the results and correct classification in individual fracture risk assessment are of great value, the present study was carried out to establish a reference database for calcaneal QUS measurements across age group and gender in Greece. A total of 1205 subjects (821 females and 384 males) from three age groups (409 children, 341 adults and 455 elderly) were recruited. QUS measurements were performed at the heel with the Sahara device, which measures broadband ultrasound attenuation (BUA) and speed of sound (SOS), and then combines these variables into a single parameter, the quantitative ultrasound index (QUI). Overall, gender-related differences were more pronounced among the elderly, while age-related differences were more pronounced among females. Elderly men had higher QUS parameters than women of peer age, but no major gender differences were observed in children and adults. In males, only BUA showed a variation with age, being higher in adult and elderly men compared to boys. On the other hand, all QUS parameters varied significantly with age in females, the general trends being mildly positive from childhood to adulthood, when peak levels were observed, and negative thereafter. The results for the Greek population were in the range reported previously for other populations, but some discrepancies were evident, probably resulting from ethno-specific characteristics and different QUS instrumentation. Importantly, using the manufacturer's or the local database as the reference population for computing T-scores led to significantly different classification of subjects into conventional categories of risk. These data could be useful as a guide for comparing the results of individual studies, as well as for the assessment of Greek men and women at risk of fracture.