More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid called seminolipid. The galactosylation of the molecule is catalyzed by UDP-galactose:ceramide galactosyltransferase (CGT). Disruption of the CGT gene in mice results in male infertility due to the arrest of spermatogenesis, indicating that seminolipid plays an important role in reproductive function. Seminolipid molecules can be assigned to different molecular species based on the fatty acid composition. In this report, we investigated the localizations of the molecular species of seminolipid by imaging mass spectrometry and demonstrated that major molecule (C16:0-alkyl-C16:0-acyl) was expressed throughout the tubules: some (C16:0-alkyl-C14:0-acyl and C14:0-alkyl-C16:0-acyl) were predominantly expressed in spermatocytes and the other (C17:0-alkyl-C16:0-acyl) was specifically expressed in spermatids and spermatozoa. This is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation.