The two-component regulatory system PhoPQ has been shown to regulate the expression of virulence factors in a number of bacterial species. For one such virulence factor, lipopolysaccharide (LPS), the PhoPQ system has been shown to regulate structural modifications in Salmonella enterica var Typhimurium. In Yersinia pestis, which expresses lipo-oligosaccharide (LOS), a PhoPQ regulatory system has been identified and an isogenic mutant constructed. To investigate potential modifications to LOS from Y. pestis, which to date has not been fully characterized, purified LOS from wild-type plague and the phoP defective mutant were analysed by mass spectrometry. Here we report the structural characterization of LOS from Y. pestis and the direct comparison of LOS from a phoP mutant. Structural modifications to lipid A, the host signalling portion of LOS, were not detected but analysis of the core revealed the expression of two distinct molecular species in wild-type LOS, differing in terminal galactose or heptose. The phoP mutant was restricted to the expression of a single molecular species, containing terminal heptose. The minimum inhibitory concentration of cationic antimicrobial peptides for the two strains was determined and compared with the wild-type: the phoP mutant was highly sensitive to polymyxin. Thus, LOS modification is under the control of the PhoPQ regulatory system and the ability to alter LOS structure may be required for survival of Y. pestis within the mammalian and/or flea host.