Horizontal gene transfer is an important evolutionary force, facilitating bacterial diversity. It is thought to be pervasive in host-associated microbiomes, where bacterial densities are high and mobile elements are frequent. These genetic exchanges are also key for the rapid dissemination of antibiotic resistance. Here, we review recent studies that have greatly extended our knowledge of the mechanisms underlying horizontal gene transfer, the ecological complexities of a network of interactions involving bacteria and their mobile elements, and the effect of host physiology on the rates of genetic exchanges. Furthermore, we discuss other, fundamental challenges in detecting and quantifying genetic exchanges in vivo, and how studies have contributed to start overcoming these challenges. We highlight the importance of integrating novel computational approaches and theoretical models with experimental methods where multiple strains and transfer elements are studied, both in vivo and in controlled conditions that mimic the intricacies of host-associated environments.