Purpose: Isolated gonadotropin-releasing hormone deficiency (IGD) is a genetically heterogeneous disorder caused by ＞30 different genes, which accounts for 30–35% of cases. Thus, Sanger sequencing is time consuming and expensive procedure and could be a challenge for etiologic diagnosis of IGD. In addition, mutations may exist in noncoding areas of the known IGD genes or still unknown genes. Recent advances in next-generation sequencing technologies have revolutionized the identification of causative genes using massively parallel sequencing of multiple samples. This study was performed to investigate the genetic etiologies of IGD using targeted sequencing of 70 known IGD genes in human. Method: This study included 29 patients with IGD from 28 independent families. Exomes were captured using customized SureSelect kit (Agilent Technologies) and sequenced on the Miseq platform (Illumina, Inc.), which includes a 163,269 bp region spanning 70 genes. Mean coverage was at least 150× for each sample, and nearly 99.9% of the targeted bases were read. We classified sequence variants into 5 main categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) according to the American College of Medical Genetics and Genomics guidelines. Results: We identified known pathogenic mutations in CHD7 gene in two males (6.8%) and PROKR2 gene in a male patient (3.4%). Novel variants were identified in 12 patients (41.4%) in BBS1, KAL1, CHD7, FGFR1, SOX3, and TACR3 genes. Of these, 7 patients (24.1%) were presumed to be pathogenic or likely pathogenic, while the remaining 5 patients had variants of uncertain significance. Two prepubertal males with anosmia were enrolled and one of them revealed novel heterozygous variant in FGFR1 gene. Conclusions: We identified genetic causes in 34.4% of patients with IGD by massively parallel sequencing using targeted gene panel. Early identification of mutations in IGD genes can provide timely hormone treatment to induce pubertal development. This approach allows for the comprehensive genetic screening in patients with IGD in a fast and cost-efficient manner. In vitro analysis is needed to verify functional implications of the novel variants.