Unusual dTDP-sugars are key intermediate in many pathogenic bacteria.  In this study, negative- ion electrospray tandem mass spectrometry (ESI-MS-MS) with collision-induced dissociation (CID) was used to study the fragmentation characteristics of six unusual nucleotide diphosphate sugars.  The results indicated the major fragment of the six unusual nucleoside sugars observed in the ESI-MS-MS spectra resulted from cleavage of diphosphate moiety and their characteristic fragment ions at m/z 401, 383, and 321, correspond to TDP−H⁻ together with fragment ions resulting from the loss of water and phosphate moiety, respectively.  Furthermore, 4-position substituted change of unusual sugar rings affected the stability of two important characteristic fragment ions of glycosyl-1''-PO₃⁻ and glycosyl-1''-P₂O₆⁻.