Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting ~1:7500 individuals characterised by recurrent chronic respiratory infections, bronchiectasis, laterality defects, and infertility. While at least 60 PCD-causing genes are reported, approximately 30% of patients remain without a confirmed molecular diagnosis. PCD caused by biallelic HYDIN variants is difficult to diagnose due to a 98% homologous pseudogene (HYDIN2). Additionally, functional ciliary diagnostic testing can be inconclusive due to the lack of discernible HYDIN ciliary ultrastructure defects by electron microscopy. Our aim was to diagnose genetically unresolved PCD patients with strong clinical suspicion of HYDIN defects using RNA sequencing (RNA-seq). RNA-seq was performed on total RNA extracted from patients' nasal epithelium cultured for 21 days at the air-liquid interface. Splicing abnormalities were detected using rMATS and genome-aligned reads were manually inspected using Integrative Genome Viewer. Thirteen individuals had strong clinical suspicion of PCD, normal TEM and inconclusive genetics: 11/13 had a single pathogenic or likely pathogenic variant in HYDIN, 1/13 had no variants, and 1/13 required clarification of variant origin (HYDIN vs HYDIN2). RNA-seq resolved 11/13 (85%) of cases. The same complex pathogenic aberrant splicing events were seen in seven unrelated patients. This abnormal splice pattern was caused by a large 10-kilobase deletion spanning intron 17 to intron 18. RNA-seq increases the diagnostic yield for patients with suspected PCD and has exposed a HYDIN 'hotspot' variant, increasing the observed prevalence of HYDIN-related cases within our genetically diagnosed cohort. Routine PCD genomics testing will now incorporate investigation of this genetic region of HYDIN.