Genetic testing of PKD1 and PKD2 is useful for diagnosis and prognosis of autosomal dominant polycystic kidney disease (ADPKD), particularly in asymptomatic individuals or those without a family history. PKD1 testing is complicated by the large transcript size, complexity of the gene region, and the extent of gene variations. A molecular assay was developed using Transgenomic's SURVEYOR Nuclease and WAVE Nucleic Acid High Sensitivity Fragment Analysis System to screen for PKD1 and PKD2 variants, followed by sequencing of variant gene segments, thereby reducing the sequencing reactions by 80%. This method was compared to complete DNA sequencing performed by a reference laboratory for 25 ADPKD patients from 22 families. The pathogenic potential of gene variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice-site alterations. A total of 90 variations were identified, including all 82 reported by the reference laboratory (100% sensitivity). A total of 76 variations (84.4%) were in PKD1 and 14 (15.6%) in PKD2. Definite pathogenic mutations (seven nonsense, four truncation, and three splicing defects) were detected in 64% (14/22) of families. The remaining 76 variants included 26 missense, 33 silent, and 17 intronic changes. Two heterozygous nonsense mutations were incorrectly determined by the reference laboratory as homozygous. "Probably pathogenic" mutations were identified in an additional five families (overall detection rate 86%). In conclusion, the SURVEYOR nuclease method was comparable to direct sequencing for detecting ADPKD mutations, achieving high sensitivity with lower cost, providing an important tool for genetic analysis of complex genes.