Titin is a giant filamentous elastic protein that spans from the Z-disk to M-band regions of the sarcomere. The I-band region of titin is extensible and develops passive force in stretched sarcomeres. This force has been implicated as a factor involved in regulating cardiac contraction. To better understand the adaptation in the extensible region of titin we report the sequence and annotation of the chicken and mouse titin genes and compare them to the human titin gene. Our results reveal a high degree of conservation within the genomic region encoding the A-band segment of titin, consistent with the structural similarity of vertebrate A-bands. In contrast the genomic region encoding the Z-disk and I-band segments is highly divergent. This is most prominent within the central I-band segment, where chicken titin has fewer but larger PEVK exons (up to 1992 bp). Furthermore, in mouse titin we found two LINE-repeats that are inserted in the Z-disk and I-band regions - the regions that account for most of the splice isoform diversity. Transcript studies show that a group of 55 I-band exons is differentially expressed in chicken titin. Consistent with a large degree of titin isoform plasticity and variation in PEVK content, chicken skeletal titins range in size from ~3000 to ~3700 kDa, and vary greatly in passive mechanical properties. Low angle X-ray diffraction experiments reveal significant differences in myofilament lattice spacing that correlate with titin isoform expression. We conclude that titin splice diversity regulates structure and biomechanics of the sarcomere.