Lipo-chitin oligosaccharides (LCOs) are novel bacterial glycolipid signal molecules that mediate the species--specific symbiosis between rhizobial bacteria and leguminous plants. Nodulation of the legume roots and nitrogen-fixation in the resulting nodules by Rhizobia is controlled by the bacterial nodulation genes that encode the LCO biosynthetic enzymes. The length of the LCO chitin backbone, the length and degree of unsaturation of the fatty acyl chain attached to it, and the combination of different chemical substituents on the reducing- and nonreducing-terminal residues all contribute to the species--specificity of the signal. LCOs are bioactive in the nanomolar and subnanomolar concentration range and are produced as heterogeneous mixtures, making determination of their structures a difficult task, most successfully approached by the application of modern mass spectrometric methods in combination with specific chemical treatments aimed at identifying specific chemical moieties. This review presents an overview of these methods as they are being used for the structural elucidation of LCOs, and discusses the role of structural diversity in mediating species-specificity.