AbstractAfter somitogenesis, skeletal muscle precursors elongate into muscle fibers that anchor to the somite boundary, which becomes the myotome boundary. Fibronectin (Fn) is a major component of the extracellular matrix in both boundaries. Although Fn is required for somitogenesis, effects of Fn disruption on subsequent muscle development are unknown. We show that fn knockdown disrupts myogenesis. Muscle morphogenesis is more disrupted in fn morphants than in a mutant where initial somite boundaries did not form, aei/deltaD. We quantified this disruption using the two‐dimensional Wavelet‐Transform Modulus Maxima method, which uses the variation of intensity in an image with respect to the direction considered to characterize the structure in a cell lattice. We show that fibers in fn morphants are less organized than in aei/deltaD mutant embryos. Fast‐ and slow‐twitch muscle lengths are also more frequently uncoupled. These data suggest that fn may function to regulate fiber organization and limit fast‐twitch muscle fiber length. Developmental Dynamics 237:2542–2553, 2008. © 2008 Wiley‐Liss, Inc.