The innate immune response depends on the ability of immune cells to detect pathogens through germline-encoded pattern recognition receptors (PRRs). Recently discovered PRRs include some members of the Pyrin and HIN domain (PYHIN) family, which are encoded on an interferon-inducible gene cluster located on chromosome 1q23. There are five human PYHIN proteins; Absent in melanoma 2 (AIM2), IFN-γ inducible protein 16 (IFI16), Myeloid cell nuclear differentiation antigen (MNDA), Pyrin and HIN domain family member 1 (PYHIN1) and the recently identified Pyrin domain only protein 3 (POP3). Early studies reported roles for these proteins in cell cycle control, tumour suppression and transcriptional regulation. AIM2 and IFI16 have now been shown to be immune sensors of non-self DNA, such as that produced by viruses in infected cells. AIM2 binds DNA to activate the inflammasome, while IFI16 detection of DNA can lead to the up-regulation of type I interferons or inflammasome activation. Recent studies have shown how IFI16 senses DNA viruses, and also how viruses evade detection by IFI16, while structural studies have greatly advanced our understanding of how AIM2 and IFI16 bind DNA to activate these immune responses. Furthermore, following the identification of POP3, interplay between members of this gene cluster has been established, with POP3 acting as a negative regulator of the AIM2 and IFI16 inflammasomes. In this review we discuss the current understanding of how PYHIN proteins function in innate immunity, their role in disease and the therapeutic possibilities that arise as a result.