Our aim was to assess reference values of thyroid volume by ultrasonography in healthy adult subjects. We conducted an epidemiological cross-sectional study where 880 subjects were randomly selected from the town census of L'Hospitalet de Llobregat after being invited to participate in our study directly by mail and phone call. We made a clinical history of each subject and determined serum thyrotropin, antiperoxidase antibodies, urinary iodine excretion and thyroid volume by ultrasonography. Subjects with thyroid disease were excluded. We finally studied 268 representative subjects. The reference thyroid volume was median 7.31 ml, mean 8.22 ml (Confidence Interval: 7.75 - 8.69 ml). In men: median 9.19 ml, mean 9.87 ml (CI: 9.09 - 10.65 ml); in women: median 6.19 ml, mean 6.57 ml (CI: 6.22 - 9.92 ml) (p < 0.0001). We grouped the subjects into decades, and found that thyroid volume was different (p = 0.0034) in males because the younger group had lower volume. We did not find any differences among age groups in women. The mean of the urinary iodine excretion was 154.23 microg/l. We have determined reference values of thyroid volume measured by ultrasonography in our iodine non-deficient population and prepared tables that distribute thyroid volume by sex and age.