Side population (SP) cells isolated from bone marrow, skeletal muscle, and skin have been shown to engraft in dystrophic muscle. However, there have been questions on the phenotypical heterogeneity, tissue of origin, and relationships among SP cell populations extracted from different tissues. Studies on bone marrow SP cells have followed a consistent protocol for their isolation and results obtained are concordant. In contrast, protocols for the isolation of muscle SP cells vary greatly, and consequently reports on their phenotype, differentiation potential and origin have been inconsistent. To address this controversy, we demonstrate that isolation parameters, such as tissue dissociation, cell counting, Hoechst concentration, and stringency in the selection of SP cells, have an effect on the yield, viability, and homogeneity of SP cells derived from bone marrow, skeletal muscle, and skin. In this paper, we demonstrate that SP cells isolated from the bone marrow are distinct from SP cells extracted from skeletal muscle and skin tissues. This study offers an explanation for the controversy surrounding muscle SP cells, provides a detailed standardized protocol for their isolation, and highlights basic guidelines for reproducible and reliable isolation of SP cells from any tissue.