Quantitative metabolomic analysis was performed for eleven tissues of freshwater fish pike-perch (Sander lucioperca), including gill, heart, liver, kidney, spleen, muscle, brain, milt, lens, aqueous (AH) and vitreous (VH) humors with the use of NMR spectroscopy. The absolute values of concentrations were determined for more than 65 most abundant metabolites in every tissue. It was found that from the metabolomic viewpoint, kidney and gill are the most similar tissues, while the metabolomic compositions of ocular tissues—lens, AH, and VH significantly differ from that of other tissues. The combinations of intracellular osmolytes and antioxidants are specific for every tissue. In particular, the concentration of antioxidant ovothiol A in the lens is much higher than in any other tissue, while the brain enjoys the elevated level of ascorbate. The most abundant osmolyte in the fish spleen, muscle, and heart is taurine, and in the brain, gill, and lens—myo-inositol. Other important osmolytes specific for particular tissues are N-acetyl-histidine, N-acetyl-aspartate, betaine, threonine-phosphoethanolamine, and serine-phosphoethanolamine. The quantitative data obtained in the present work can be used as the baseline metabolite concentrations in the fish tissues to evaluate the influence of seasonal, ecological and other factors on the fish metabolism.