AbstractMass spectrometry‐based shotgun lipidomics was applied to the analysis of sphingolipids of 11 yeast strains belonging to four genera, that is Cryptococcus, Saccharomyces, Schizosaccharomyces, and Wickerhamomyces. The analysis yielded comprehensive results on both qualitative and quantitative representation of complex sphingolipids of three classes—phosphoinositol ceramide (PtdInsCer), mannosyl inositol phosphoceramide (MInsPCer), and mannosyl diinositol phosphoceramide (M(InsP)2Cer). In total, nearly 150 molecular species of complex sphingolipids were identified. Individual strains were cultured at five different temperatures, that is 5, 10, 20, 30, and 40 °C (Wickerhamomyces genus only up to 30 °C), and the change in the culture temperature was found to affect the representation of both the sphingolipid classes and the length of the long‐chain bases (LCB). Individual classes of sphingolipids differing in polar heads differed in the temperature response. The relative content of PtdInsCer increased with increasing temperature, whereas that of M(InsP)2Cer decreased. Molecular species having C18‐LCB were associated with low cultivation temperature, and a higher proportion of C20‐LCB molecular species was produced at higher temperatures regardless of the type of polar head. On the other hand, the influence of temperature on the representation of very long‐chain fatty acids (VLCFA) was less noticeable, the effect of the taxonomic affiliation of the strains being more pronounced than the cultivation temperature. For example, lignoceric and 2‐hydrocylo‐lignoceric acids were characteristic of the genera Cryptococcus and Schizosaccharomyces, and of Saccharomyces genus cultivated at high temperatures.