Background: Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disorder characterized by bone loss and bone fragility. The aim of this study was to investigate the variants of three genes involved in the pathogenesis of OI. Methods: Molecular genetic analyses were performed for COL1A1, COL1A2, and CRTAP genes in an Iranian family with OI. The DNA samples were analyzed by next-generation sequencing (NGS) gene panel and Sanger sequencing. Results: Five different variants were identified in COL1A1 and COL1A2, including two variants in COL1A1 and three variants in COL1A2. Among the five causative COL1A1 and COL1A2 variants, one novel variants, c.1081 G>A, was found in COL1A2, which was identified in two siblings. Conclusion: Our finding extends the variant spectrum of the COL1A2 gene and has important implications for genetic counseling of families. The NGS is a powerful molecular diagnostic strategy for OI, a heterogeneous disorder.