Skeletal muscles are not created equal. The underutilized concept of muscle allotypes defines distinct muscle groups that differ in their intrinsic capacity to express novel traits when exposed to a facilitating extrinsic environment. Allotype-specific traits may have significance as determinants of the preferential involvement or sparing of muscle groups that is observed in a variety of neuromuscular diseases. Little is known, however, of the developmental mechanisms underlying the distinctive skeletal muscle allotypes. The lack of appropriate in vitro models, to dissociate the cell-autonomous and non-cell-autonomous mechanisms behind allotype diversity, has been a barrier to such studies. Here, we derived novel cell lines from the extraocular and hindlimb muscle allotypes and assessed their similarities and differences during early myogenesis using morphological and gene/protein expression profiling tools. Our data establish that there are fundamental differences in the transcriptional and cellular signaling pathways used by the two myoblast lineages. Taken together, these data show that myoblast lineage plays a significant role in the divergence of the distinctive muscle groups or allotypes.