Bacteriophages (phages) are viruses that infect bacteria, and they have been evolving for billions of years to combat the hosts they infect as well as other phages in the environment. Many phages are temperate, and after infection they may remain in the host as a prophage, forming a lysogen instead of initiating lytic growth. Lysogeny presents unique challenges and benefits, and as a result temperate phages impact their environment differently than obligately lytic phages. Although temperate phages are diverse, many paradigms about their lifestyle and evolution have been derived from small collections of phages representing limited and ill-defined genetic diversity. Therefore, I have investigated a large collection of phages infecting hosts in the phylum Actinobacteria to expand our understanding of temperate phage diversity and evolution. First, I show that in contrast to obligately lytic phages, temperate phages evolve within two evolutionary modes that are characterized by different degrees of gene content flux and that vary by the type of temperate phage and its bacterial host. Second, I characterize extrachromosomal Mycobacterium prophages that utilize partitioning systems to maintain lysogeny, which are not commonly reported. I show that these prophages exhibit partition-mediated incompatibility and that components of this system evolve under different selective pressures possibly to avoid this instability. Third, I characterize Bifidobacterium prophages and explore how they interact with their hosts. Some of these prophages utilize a unique integration site and encode a shufflon system. This shufflon may control host range and is the first to be reported in actinobacteriophages. Last, I examine the evolution of a Mycobacterium prophage immunity system. This regulatory circuit enables prophages to control expression of lytic genes, maintaining lysogeny and defending against superinfection. I show that closely-related phages with diverging immunity systems generate a complex immunity network and gaining virulence to escape this network is difficult. Overall, this research has broadened our understanding of temperate phage diversity and evolution, and it has identified genetic systems that can be used to develop new genetic tools
