Background: Vertebrate muscles are defined and patterned at the stage of primary myotube formation, but there is no clear description of how these cells form in vivo. Of particular interest is whether primary myotubes are “seeded” by a unique myoblast population that differentiates as mononucleated myocytes, similar to the founder myoblasts of insects. Results: We analyzed the cell populations and processes leading to initiation of primary myogenesis in limb buds of rats and mice. Pax3+ve myogenic precursors migrate into the limb bud and initially consolidate into dorsal and ventral muscle masses in the absence of Pax7 expression. Approximately a day later, Pax7+ve cells appear in the central aspect of the limb base and subsequently throughout the limb muscle masses. Primary myogenesis is initiated within each muscle mass at a time when only Pax3, and not Pax7, protein can be detected. Primary myotubes form initially as elongate mononucleated myocytes, well before cleavage of the muscle masses has occurred. Multinucleate myotubes appear approximately a day later. A similar process is seen during initiation of chick limb primary myogenesis. Conclusions: Primary myotubes of vertebrate limb muscles are initiated by mononucleated myocytes, that appear structurally analogous to the founder myoblasts of insects. Developmental Dynamics 242:1043–1055, 2013. © 2013 Wiley Periodicals, Inc.