Objective Thyroglobulin (Tg) is an important tumor marker for therapy control and follow-up of differentiated thyroid carcinoma (DTC). Over the past decade, assays for highly sensitive Tg measurement have become increasingly established. We evaluated a newly developed high-sensitive Tg assay running on an automated platform (LIAISON® Tg II Gen assay, DiaSorin), with a limit of quantification of 0.10 ng/ml. Design and Methods Tg values of 166 sera from subjects without thyroid diseases and of more than 500 sera of well-defined DTC patients were determined with the new LIAISON® Tg II Gen assay and compared with two established assays (Elecsys® Tg II/Roche, and Medizym® Tg REM/Medipan). Results Tg reference values from healthy subjects were up to 37.93 ng/ml (women) resp. 24.59 ng/ml (men) with the LIAISON® Tg II Gen assay. Tg values showed good correlations in healthy subjects and patients with active tumorous disease. In contrast, Tg values in the very low range from cured thyroidectomized patients were poorly comparable between the three assays, while clinical differences between the cohorts were correctly reflected by all assays. Conclusions With the new LIAISON® Tg II Gen assay, another automated assay standardized against the first International Reference Preparation CRM-457 for highly sensitive measurement of Tg values is available.