Background <p>Cystic kidney disease is a heterogeneous chronic kidney disease. It is often difficult to make a definitive diagnosis based on clinical and ultrasound features alone.</p> Methods <p>A total of 40 families with cystic kidney disease from Southwest China to meet their genetic counseling and prenatal diagnosis needs. To detect renal disease genes, NGS was performed on probands with cystic kidney disease, and candidate variants were validated using Sanger sequencing.</p> Results <p>Among the 40 families, a definitive genetic diagnosis was established in 30 cases (30/40, 75.0%), with seven patients having family members who subsequently underwent prenatal diagnosis based on these molecular findings. We identified a total of 38 variants. According to the criteria of the American College of Medical Genetics and Genomics, 31 variants were classified as pathogenic or likely pathogenic and sevent were classified as uncertain significance. Of the 38 variants, 27 were known and 11 were novel.</p> Conclusions <p>Our study expands the mutation spectrum for cystic kidney disease gene. Genetic testing is important for clinicians diagnose and guide treatment patients with different types of cystic kidney disease accurately. NGS is a detection technology that can be considered, which is effective in avoiding unexpected misdiagnoses due to clinical overlap among cystic kidney disease. Finally, these results may be useful for the differential diagnosis and genetic counseling of patients with cystic kidney disease.</p>