Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37°C, Y. pestis lipopolysaccharide (LPS) contained the tetra‐acylated lipid IV_A and smaller amounts of lipid IV_A modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IV_A modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra‐acylated lipid A. When grown at 21°C, however, the three yersiniae synthesized LPS containing predominantly hexa‐acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor‐α, whereas the lipid A synthesized by the three species at 37°C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature‐dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species‐specific lipid A forms may be important for life cycle and pathogenicity differences.