Mitochondria are believed to play an important role in shaping the intracellular Ca2+ transients during skeletal muscle contraction. There is discussion about whether mitochondrial matrix Ca2+ dynamics always mirror the cytoplasmic changes and whether this happens in vivo in whole organisms. In this study, we characterized cytosolic and mitochondrial Ca2+ signals during spontaneous skeletal muscle contractions in zebrafish embryos expressing bioluminescent GFP-aequorin (GA, cytoplasm) and mitoGFP-aequorin (mitoGA, trapped in the mitochondrial matrix). The Ca2+ transients measured with GA and mitoGA reflected contractions of the trunk observed by transmitted light. The mitochondrial uncoupler FCCP and the inhibitor of the mitochondrial calcium uniporter (MCU), DS16570511, abolished mitochondrial Ca2+ transients whereas they increased the frequency of cytosolic Ca2+ transients and muscle contractions, confirming the subcellular localization of mitoGA. Mitochondrial Ca2+ dynamics were also determined with mitoGA and were found to follow closely cytoplasmic changes, with a slower decay. Cytoplasmic Ca2+ kinetics and propagation along the trunk and tail were characterized with GA and with the genetically encoded fluorescent Ca2+ indicator, Twitch-4. Although fluorescence provided a better spatio-temporal resolution, GA was able to resolve the same kinetic parameters while allowing continuous measurements for hours.