Synechocystis strains are considered as valuable resources for production of useful metabolites. Comparative primary metabolic and lipidomic profiling of freshwater and marine Synechocystis strains is needed to understand the physiological characteristics of each strain at molecular level. In this study, we performed comparative primary metabolic and lipidomic profiling of freshwater Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) and marine Synechocystis sp. PCC 7338 (hereafter Synechocystis 7338) by using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry. Neophytadiene, phytol, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) were significantly higher in Synechocystis 6803. Levels of alanine, aspartic acid, valine, linoleic acid, linolenic acid, oleic acid, plamitic acid, stearic acid, glucosylglycerol, sucrose, phosphatidylglycerol (PG), and diacylglyceryltrimethylhomoserine (DGTS) (only detected in Synechocystis 7338) were significantly higher in Synechocystis 7338. Our results provide basic information to choose Synechocystis strains for various industrial usages, and also provide fundamental information on the future development of novel Synechocystis strains to produce high levels of specific metabolites by metabolic pathway modulation.