Transposable elements (TEs) are mobile genetic elements that can threaten genome stability and disrupt reproduction. PIWI-interacting RNAs (piRNAs) are a specialized small RNAs that silence TEs in the animal germline. In my dissertation, I explore both the evolutionary and functional aspects of the PIWI-piRNA pathway using two model systems: Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish). piRNAs are produced from discrete genomic regions called piRNA clusters. Although the molecular processes by which piRNA cluster’s function are extensively studied in Drosophila melanogaster, much less is known about the origin of piRNA clusters in this or any other species. In Chapter 3, I investigated the origin and evolution of piRNA clusters. My analysis uncovers extensive variation in piRNA cluster activity across strains, and signatures of rapid birth and death of such loci. Patterns of piRNA cluster variation in activity and sequence composition indicate TEs themselves are the major force driving the rapid evolution of piRNA clusters.Among vertebrates, functional understanding of PIWIs and piRNAs is mainly derived from mice. Zebrafish offers a unique opportunity to study the piRNA pathway in the context of a vertebrate genome with a rich diversity of TEs. However, direct targets of the zebrafish PIWI-piRNA pathway have remained uncharacterized. In Chapter 3, I provide a comprehensive view of the targets of the zebrafish piRNA pathway. I identified that silencing of most targets of the piRNA pathway is piwi-dose dependent. I hypothesize that this unexpected repression effect is the result of ectopic activation of other silencing pathway(s). Notably, we find that ‘Fish N-terminal zinc finger’ (FiNZ) domain encoding genes, which are thought to transcriptionally repress TEs, are upregulated in piwi mutant gonads. Since FiNZ-ZNFs also silence TEs but through chromatin modifications, such a crosstalk between PIWI-piRNAs and FiNZ-ZNFs may dictate rules of hierarchy and balance in TE regulation. Together, these studies highlight the dynamic nature of the piRNA pathway across short and long-term evolution. By combining evolutionary genomics with functional analysis in multiple model organisms, my dissertation sheds light on the conserved logic and species-specific dynamics of the PIWI-piRNA system.