The zebrafish thyroid gland shows a unique pattern of growth as a differentiated endocrine gland. Here, we analyze the onset of differentiation, the contribution of lineages, and the mode of growth of this gland. The expression of genes involved in hormone production and the establishment of epithelial polarity show that differentiation into a first thyroid follicle takes place early during embryonic development. Thyroid follicular tissue then grows along the pharyngeal midline, initially independently of thyroid stimulating hormone. Lineage analysis reveals that thyroid follicle cells are exclusively recruited from the pharyngeal endoderm. The ultimobranchial bodies that merge with the thyroid in mammals form separate glands in zebrafish as visualized by calcitonin precursor gene expression. Mosaic analysis suggests that the first thyroid follicle differentiating at 55 hours postfertilization corresponds later to the most anterior follicle and that new follicles are added caudally. Developmental Dynamics 235:1872–1883, 2006. © 2006 Wiley-Liss, Inc.