Mammals harbor hundreds of bacterial species in the gut that are deeply integrated with their hosts’ metabolic, immune, and neuroendocrine systems. Before birth, mammals lack a defined gut microbiota, which must be assembled anew in each host generation. Individuals acquire their first inoculum from the mother during birth and are subsequently colonized throughout life by bacteria from the external environment, including social contacts. However, the modes by which specific gut bacterial lineages are transmitted between hosts remain poorly understood. It currently remains unknown which, if any, gut bacterial lineages are faithfully inherited within mammalian host lineages over multiple generations. Similarly, the relative contributions of horizontal transmission through social interactions and shared environments are unclear. Furthermore, the underlying genetic bases of bacterial transmission phenotypes have not been discovered. Resolving these knowledge gaps is of critical significance for understanding the full complement of genetic material inherited within mammalian lineages, the evolution of symbiosis between gut bacteria and mammalian species, the community assembly of the gut microbiota within individual mammals, and the spread of bacterial enteropathogens within mammalian populations. Here, we propose to identify vertically and horizontally transmitted members of the mouse gut microbiota (Aim 1) and to discover the genetic bases of gut bacterial transmission phenotypes (Aim 2). In Aim 1, we will employ an innovative experimental system that utilizes wild-derived outbred populations of mice reared in outdoor enclosures to disentangle the modes of transmission within the gut microbiota. As part of this work, we will also develop new approaches for assembling high-quality bacterial genomes from metagenomes that will provide unprecedented opportunities to study dispersal of bacteria. In Aim 2, we will employ transposon- insertion sequencing of gut bacterial lineages that disperse within and between germ-free mouse lines reared in gnotobiotic isolators in order to identify the specific genes that underlie gut bacterial transmission phenotypes. This work will focus on both vertically and horizontally transmitted bacterial lineages identified by our previous work as well as by results of Aim 1. Determining the genetic basis of gut bacterial transmission within and between mammalian lineages has the potential to reshape understanding of the mechanisms and evolution of bacterial dispersal strategies. In addition, Aim 2 will contribute substantially to the development of functional genomics tools in mammalian gut bacteria. Cumulatively, the proposed work will yield fundamental insights into the modes of gut bacterial transmission in mammals.