Background <p>Few epidemiological evidence is available on genetic hypoparathyroidism (HP) in adult-onset non-surgical hypoparathyroidism (ns-HP). This study aimed to investigate the spectrum of genotypes and clinical phenotypes of genetic HP in a single-center large sample of Chinese adult-onset ns-HP cohort.</p> Methods <p>A systemic screening of HP-causing genes and clinical studies were conducted in adult-onset (age &gt; 18 years old) ns-HP patients. Targeted next-generation sequencing/whole exome sequencing (T-NGS/WES) and multiplex ligation-dependent probe amplification (MLPA) of the <i>TBX1</i> gene were performed to identify rare variants of 20 HP-causative genes. The fluorescence imaging of intracellular ionized calcium (Ca²⁺) experiments were conducted to identify the function of <i>CASR</i> with variants of uncertain significance. Clinical data were collected retrospectively and compared between patients with pathogenic/likely pathogenic (P/LP) variants and idiopathic HP (IHP).</p> Results <p>A total of 97 adult-onset ns-HP patients were enrolled in the study. The detection rate of P/LP variants was 5.2% (5/97). Five HP patients were identified to carry five P/LP rare variants of four genes, including <i>GATA3</i>, <i>TBX1</i> (<i>n</i> = 2), <i>CASR</i>, and <i>AIRE</i>. Three variants were newly identified in our study. The mean onset age of the five patients with P/LP variants was 37.6 ± 14.5 years. No significant differences in terms of most clinical characteristics between the P/LP group and the IHP group were found in our study.</p> Conclusions <p>Genetic factors may not be the main cause of ns-HP, and it is better to screen the common causative genes including <i>TBX1</i>, <i>CASR</i>, <i>AIRE</i>, and <i>GATA3</i> in adult-onset ns-HP patients with a positive family history, syndromic phenotype, trend to hypercalciuria, or other hereditary signs.</p>