Pathogenic species of the genus Yersinia have a marked tropism for lymphoid tissue during the early stages of infection. Bacterial survival at this site determines whether the disease is localized or progresses systemically, leading to a high rate of mortality. Several plasmid‐encoded virulence genes are known to be required for survival and pathogenesis, but the contribution of chromosomal genes has been largely unexplored. This study represents the first intensive effort to characterize and determine the function of Yersinia chromosomal genes expressed in lymphoid tissue after intragastric infection. Strains harbouring cat fusions expressed in the host were isolated from Peyer’s patch tissue of mice intragastrically infected and treated with chloramphenicol (Cm); genes identified in this manner were designated hre for host responsive element. The hre::cat strains that were Cm resistant in vivo (in mouse tissue) and Cm sensitive in vitro (on laboratory media at 26°C) were identified and shown to consist of 61 different allelic groups. The hre::cat fusions from 48 of the allelic groups were cloned and characterized by DNA sequence analysis. The results identified genes necessary for iron acquisition, protection from environmental stresses, biosynthesis of cell envelope components and other diverse metabolic activities. However, the DNA sequence of many clones had no homology to other known genes. Insertion mutations were constructed for four hre genes and the resulting Y. enterocolitica mutants were tested in the mouse model for effects on pathogenesis. All of the mutant strains were affected for virulence when assayed for survival in host tissues and LD50 analysis.