Gangliosides, a subtype of glycosphingolipids containing sialic acid, play important roles in various biological processes such as cell-cell recognition and interaction, and modulation of membrane protein function. In particular, it has attracted considerable attention for a long time due to their close association with cancer pathology such as modified expression patterns in cancer cells related to apoptosis resistance. Although the important biological functions of the gangliosides continue to be revealed, there is only few study on comprehensive profiling of gangliosides due to the lack of effective analytical method for proving their structural complexity (hydrophobic ceramide moiety and hydrophilic glycan chain). In this study, we performed profiling and structural elucidation of gangliosides in the different cancer cell lines such as CFPAC1, A549, NCI-H358, MCF7, and Caski using negative ion detection mode nano LC/MS and MS/MS. Extracted gangliosides were assigned based on accurate mass, known glycobiology, and established mathematical model, and then the structures were further elucidated using tandem MS, retention time. In total, over 70 ganglioside compositions were identified from 5 cancer cell lines. Notably, the different cell lines were obviously distinguished through the difference of relative abundance of various gangliosides. In particular, the distribution of glycan moiety was more distinctly differed in each cell line than the ceramide moiety between different cell lines. These results demonstrated that cancer origins and molecular subtypes could be definitely differentiated based on distinctive ganglioside profiles. This result shows the feasibility of cell surface ganglioside as effective targets for diagnosis of cancer cell types.