Abstract Most muscle originates from the myotomal compartment of the somites, paired structures flanking the neural tube. Whereas vertebrate embryos show molecular and morphological asymmetry about the left–right body axis, somitic myogenesis is thought to occur symmetrically. Here, we provide the first evidence that myotome pairs are transiently left–right asymmetric, with higher expression of α‐ skeletal actin and myosin light chain 3F ( MLC3F ) on the left side between embryonic day 9.5–10.25. In iv mutants with situs inversus , the asymmetric expression of α‐ skeletal actin and MLC3F was inverted, showing that this process is regulated by global left–right axis cues, initiated before gastrulation. However, although left–sided identity is later maintained by Pitx2 genes, we found that Pitx2c null embryos have normal left‐biased expression of α‐ skeletal actin and MLC3F . Myotome asymmetry, therefore, is downstream of the iv mutation but upstream of, or unrelated to, the Pitx2c pathway. Developmental Dynamics 231:795–800, 2004. © 2004 Wiley‐Liss, Inc.