Piwis are regulatory proteins that belong to the Argonaute gene family. Piwis are known to defend host genomes against transposable element (TE) mobilization in germline cells and use a class of small RNA, piRNA, as guides to TE transcripts and loci. The Argonaute family has members among all eukaryotes, but Piwis are only present in animals. Piwis are primarily expressed in gonadal tissues and protect inheritable genomes against the mobilization and propagation of TEs through transcript cleavage and TE methylation. Vertebrates genomes encode up to four Piwis named Piwil1, Piwil2, Piwil3 and Piwil4. Piwil1 and Piwil2 have been described in most animals, but Piwil3 and Piwil4 are specific to vertebrates and their duplication history is unresolved. Therefore, we leveraged phylogenetic, synteny and expression analyses to address this void. We collected Piwi DNA sequences from Ensembl 101 and NCBI from species representing all major vertebrate lineages. Piwi sequences were aligned and we constructed phylogenetic trees using IQTREE and ExaBayes. Our phylogenetic analysis suggests Piwil1 and Piwil2 were retained in all vertebrate members. Piwil4 was the result of Piwil1 duplication in the ancestor of gnathostomes, but was independently lost in ray-finned fishes and birds. Further, Piwil3 was also derived from a tandem Piwil1 duplicate in the common ancestor of marsupial and placental mammals. However, Piwil3 was secondarily lost in Afrotherians, Xenarthrans and mouse-like rodents. Interestingly, the evolutionary rate of Piwil3 is considerably faster than Piwil1, consistent with gene duplication models that predict relaxation of functional constraint and subsequent neofunctionalization. Further, several amino acid sites throughout the entirety of Piwil3 were evolving in a manner consistent with positive selection. Generally, gene order Piwil1, Piwil2, and Piwil4 has been conserved through vertebrate evolution, even around the Piwil4 locus in birds and ray-finned fish that lost Piwil4. The position of Piwil3 has been less conserved during mammalian evolution and can be bordered by a diversity of genes. Lastly, our expression analyses suggest Piwi expression has mostly been constrained to gonads throughout vertebrate evolution, consistent with previous analyses. Vertebrate evolution is marked by two rounds of whole genome duplication and many multigene families can be linked to this event. However, our analyses suggest Piwi duplications were independent of these events. Further, teleost fishes lack any additional Piwi paralogs despite an independent round of whole genome duplication. The loss of Piwil4 in birds and ray-finned fishes is interesting because Piwil4 is linked to preventing TE mobilization through DNA methylation. TEs are silenced at CpG sites in mammals but CpG islands are largely un-methylated in birds. It is hypothesized methylating TEs creates a non-lethal relationship between TEs and host genomes by allowing the retention of insertions that would otherwise be purged by natural selection. Piwil4 is present in other reptiles and reptilian TEs are not methylated at CpG sites, however a there could be a possible link between the loss of Piwil4 and TE reduction in birds worthy of further investigation.