The genetic study of titin has been notoriously difficult because of its size and complicated alternative splicing routes. Here, we have used zebrafish as an animal model to investigate the functions of individual titin isoforms. We identified 2 titin orthologs in zebrafish, ttna and ttnb, and annotated the full-length genomic sequences for both genes. We found that ttna, but not ttnb, is required for sarcomere assembly in the heart as well as the subsequent establishment of cardiac contractility. In fact, ttna is the earliest sarcomeric mRNA that is expressed in the heart, which makes it an early molecular marker for cardiomyocyte differentiation. Surprisingly, ttna is required for later steps of sarcomere assembly, including the assembly of Z-discs and A-bands, but not for early steps such as the assembly of Z-bodies and nonstriated myosin filaments. Reduction of individual titin isoforms in vivo using morpholino-modified antisense oligonucleotides indicated that (1) both N2B exon–containing and N2A exon–containing isoforms of ttna are required for sarcomere assembly in the heart; (2) N2A exon–containing isoforms of both ttna and ttnb are required for sarcomere assembly in the somites; and (3) the N2B exon–containing isoforms of ttnb are expressed later than other titin isoforms and are probably involved in modulating their expression; however, these isoforms of ttnb are not required for sarcomere assembly. Collectively, our results reveal distinct functions of different titin isoforms and suggest that various phenotypes in “titinopathies” may be attributable to the disruption of different titin isoforms.