The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. Five sugar nucleotides, GDP-alpha-D-mannose, UDP-alpha-D-N-acetylglucosamine, UDP-alpha-D-glucose, UDP-alpha-galactopyranose, and GDP-beta-L-fucose, were common to all three species; one, UDP-alpha-D-galactofuranose, was common to T. cruzi and L. major; three, UDP-beta-L-rhamnopyranose, UDP-alpha-D-xylose, and UDP-alpha-D-glucuronic acid, were found only in T. cruzi; and one, GDP-alpha-D-arabinopyranose, was found only in L. major. The estimated demands for each monosaccharide suggest that sugar nucleotide pools are turned over at very different rates, from seconds to hours. The sugar nucleotide survey, together with a review of the literature, was used to define the routes to these important metabolites and to annotate relevant genes in the trypanosomatid genomes.