Premature ovarian insufficiency (POI), affecting up to 1 in 100 women, is defined by amenorrhea and elevated follicle stimulating hormone before the age of 40. Causative variants have been described in more than 50 genes, but these explain only a minority, suggesting the involvement of many unidentified genes. POI-related genes affect various processes such as gonadal development, DNA replication/repair, hormonal signalling, immune function and metabolism. We have used massively parallel sequencing (MPS) to investigate the genetic basis of POI. In two individuals, we identified C-terminal truncating variants in TP63 as a new genetic cause of POI. This established a new genotype:phenotype correlation for the TP63-related syndromes. We also discovered a surprising cause of “isolated” POI in two cases. In one patient, a homozygous nonsense variant in NBN was causative. Recessive pathogenic NBN variants typically cause POI in the context of Nijmegen Breakage Syndrome, characterized by microcephaly, cancer predisposition and immunodeficiency, none of which were evident. At a cellular level, however, we found evidence of chromosomal instability, suggesting an elevated cancer risk. In the second case, compound heterozygous variants in EIF2B2 were causative. Recessive pathogenic EIF2B2 variants can cause POI in addition to progressive leukoencephalopathy. MRI revealed sub-clinical neurological abnormalities, implicating likely future decline. These diagnoses demonstrate that causative variants in pleiotropic POI genes can be identified before full clinical manifestation. As MPS is increasingly used in research and the clinic, similar cases are likely to arise at an accelerating rate. Clinicians and researchers offering MPS to patients with POI, need to understand and communicate the possible implications of diagnosis, and ideally involve genetic counsellors in patient care. Analysis of our MPS data is ongoing but includes additional diagnoses in known POI genes, as well as many variants of interest in candidate POI genes.