The physical sites of calcium entry and exit in the skeletal muscle cell are distinct and highly organised in space. It was investigated whether the highly structured spatial organisation of sites of Ca2þ release, uptake and action in skeletal muscle cells substantially impacts the dynamics of cytosolic Ca2þ handling and thereby the physiology of the cell. Hereto, the spatiotemporal dynamics of the free calcium distribution in a fast-twitch (FT) muscle sarcomere was studied using a reaction–diffusion computational model for two genotypes with known anatomical differences. A computational model of a murine FT muscle sarcomere is developed, de novo including a closed calcium mass balance to simulate spatiotemporal high stimulation frequency calcium dynamics at 358C. Literature data on high-frequency calcium dye measurements were used as a first step towards model validation. The murine and amphibian sarcomere models were phenotypically distinct to capture known differences in positions of troponin C, actin–myosin overlap and calcium release within the sarcomere between frog and mouse. The models predicted large calcium gradients throughout the myoplasm as well as differences in calcium concentrations near the mitochondria of frog and mouse. Furthermore, the predicted Ca2þ concentration was high at positions where Ca2þ has a regulatory function, close to the mitochondria and troponin C.