BACKGROUND: Rapid diagnostic whole-genome sequencing has been explored in critically ill newborns, hoping to improve their clinical care and replace time-consuming and/or invasive diagnostic testing. A previous retrospective study in a research setting showed promising results with diagnoses in 57%, but patients were highly selected for known and likely Mendelian disorders. The aim of our prospective study was to assess the speed and yield of rapid targeted genomic diagnostics for clinical application. METHODS: We included 23 critically ill children younger than 12 months in ICUs over a period of 2 years. A quick diagnosis could not be made after routine clinical evaluation and diagnostics. Targeted analysis of 3426 known disease genes was performed by using whole-genome sequencing data. We measured diagnostic yield, turnaround times, and clinical consequences. RESULTS: A genetic diagnosis was obtained in 7 patients (30%), with a median turnaround time of 12 days (ranging from 5 to 23 days). We identified compound heterozygous mutations in the EPG5 gene (Vici syndrome), the RMND1 gene (combined oxidative phosphorylation deficiency-11), and the EIF2B5 gene (vanishing white matter), and homozygous mutations in the KLHL41 gene (nemaline myopathy), the GFER gene (progressive mitochondrial myopathy), and the GLB1 gene (GM1-gangliosidosis). In addition, a 1p36.33p36.32 microdeletion was detected in a child with cardiomyopathy. CONCLUSIONS: Rapid targeted genomics combined with copy number variant detection adds important value in the neonatal and pediatric intensive care setting. It led to a fast diagnosis in 30% of critically ill children for whom the routine clinical workup was unsuccessful.