Troponin T (Tnnt), a troponin component, interacts with tropomyosin and is crucial to the regulation of striated muscle contraction. To gain insight into the molecular evolution and developmental regulation of Tnnt gene (Tnnt) in lower vertebrates, zebrafish Tnnt1 (slow Tnnt), Tnnt2 (cardiac Tnnt), and Tnnt3b (fast Tnnt isoform b) were characterized. The polypeptides of zebrafish Tnnt1, Tnnt2, and Tnnt3b were conserved in the central tropomyosin- and C-terminal troponin I-binding domains. However, the N-terminal hypervariable regions were highly extended and rich in glutamic acid in polypeptides of Tnnt1 and Tnnt2, but not Tnnt3b. The Tnnt2 and Tnnt3b contain introns, whereas Tnnt1 is intron-free. During development, large to small, alternatively spliced variants were detected in Tnnt2, but not in Tnnt1 or Tnnt3. Whole-mount in situ hybridization showed zebrafish Tnnt1 and Tnnt2 are activated during early somitogenesis (10 hr postfertilization, hpf) and cardiogenesis (14 hpf), respectively, but Tnnt3b is not activated until middle somitogenesis (18 hpf). Tnnt2 and Tnnt3b expression was cardiac- and fast-muscle specific, but Tnnt1 was expressed in both slow and fast muscles. We propose that three, distinct, muscle-type Tnnt evolved after the divergence of fish and deuterostome invertebrates. In zebrafish, the developmental regulation of Tnnt during somitogenesis and cardiogenesis is more restricted and simpler than in tetrapods. These new findings may provide insight into the developmental regulation and molecular evolution of vertebrate Tnnt. Developmental Dynamics 227:266–279, 2003. © 2003 Wiley-Liss, Inc.