The roles of sphingolipids in polycystic ovary syndrome (PCOS) are still unknown. This study aimed to investigate the sphingolipid characteristics for different types of PCOS using liquid chromatography-mass spectrometry (LC-MS). A total of 107 women with PCOS and 37 healthy women as normal controls were studied. PCOS patients were further classified into non-obesity with insulin resistance (IR) (NOIR), obesity with IR (OIR), and non-obesity and non-IR (NIR) subgroups. A total of 87 serum sphingolipids, including 9 sphingosines, 3 sphinganines, 1 sphingosine-1-phosphate (S1P), 19 ceramides (Cers), 1 ceramide-1-phosphate, 44 sphingomyelins (SMs), 4 hexosylceramides, and 6 lactosylceramides (LacCers) were analyzed using an improved sphingolipidomic approach based on LC-MS. Notable elevations in the levels of S1P, Cer, and SM were observed in PCOS patients when compared with healthy women, and SM species with long saturated acyl chains showed potential as novel biomarkers of PCOS. In addition, the level of LacCer was only elevated in NIR, and there was almost no change in NOIR and OIR. This study is the first to report the comprehensive sphingolipidomic profiling of different subgroups of PCOS with or without IR or obesity and suggests that serum sphingolipids might be useful as diagnostic biomarkers for different types of PCOS.