Myonuclei transcriptionally regulate muscle fibers during homeostasis and adaptation to exercise. Their cellular location and quantity are important when characterising phenotypes of myopathies, the effect of treatments and to understand the roles of satellite cells in muscle adaptation and muscle ‘memory’. Difficulties arise in identifying myonuclei due to their proximity to the sarcolemma and closely residing interstitial cell neighbours. We aimed to determine to what extent PCM1 is a specific marker of myonuclei in-vitro and in-vivo. Single isolated myofibers and cross-sections sections from mice and humans were studied from several models including Wild-type and Lamin A/C mutant mice after functional overload, and damage and recovery in humans following forced eccentric contractions. Fibers were immuno-labelled for PCM1, Pax7 and DNA. C2C12 myoblasts were also studied to investigate changes in PCM1 localisation during myogenesis. PCM1 labelled the nuclear envelope of myonuclei in mature myofibers and in newly formed myotubes, but also labelled centrosomes in proliferating myogenic precursors which may or may not fuse to join the myofiber syncytium. It also labelled non-myogenic nuclei near the sarcolemma especially in regenerating areas of the Lmna+/ΔK32 mouse and damaged human muscle. While PCM1 is not completely specific to myonuclei, the impact that PCM1+ macrophages and interstitial cells have on myonuclei counts would be small in healthy muscle. PCM1 may prove useful as a marker of satellite cell dynamics due to the distinct change in localisation during differentiation, revealing satellite cells in their quiescent (PCM1-), proliferating (PCM1+ centrosome), and pre-fusion states (PCM1+ nuclear envelope).