CONSPECTUS: The unique physical properties of two-dimensional (2D) molybdenum disulfide (MoS2) and its promising applications in future optoelectronics have motivated an extensive study of its physical properties. However, a major limiting factor in investigation of 2D MoS2 is its large area and high quality preparation. The existence of various types of defects in MoS2 also makes the characterization of defect types and the understanding of their roles in the physical properties of this material of critical importance. In this Account, we review the progress in the development of synthetic approaches for preparation of 2D MoS2 and the understanding of the role of defects in its electronic and optical properties. We first examine our research efforts in understanding exfoliation, direct sulfurization, and chemical vapor deposition (CVD) of MoS2 monolayers as main approaches for preparation of such atomic layers. Recognizing that a natural consequence of the synthetic approaches is the addition of sources of defects, we initially focus on identifying these imperfections with intrinsic and extrinsic origins in CVD MoS2. We reveal the predominant types of point and grain boundary defects in the crystal structure of polycrystalline MoS2 using transmission electron microscopy (TEM) and understand how they modify the electronic band structure of this material using first-principles-calculations. Our observations and calculations reveal the main types of vacancy defects, substitutional defects, and dislocation cores at the grain boundaries (GBs) of MoS2. Since the sources of defects in two-dimensional atomic layers can, in principle, be controlled and studied with more precision compared with their bulk counterparts, understanding their roles in the physical properties of this material may provide opportunities for changing their properties. Therefore, we next examine the general electronic properties of single-crystalline 2D MoS2 and study the role of GBs in the electrical transport and photoluminescence properties of its polycrystalline counterparts. These results reveal the important role played by point defects and GBs in affecting charge carrier mobility and excitonic properties of these atomic layers. In addition to the intrinsic defects, growth process induced substrate impurities and strain induced band structure perturbations are revealed as major sources of disorder in CVD grown 2D MoS2. We further explore substrate defects for modification and control of electronic and optical properties of 2D MoS2 through interface engineering. Self-assembled monolayer based interface modification, as a versatile technique adaptable to different conventional and flexible substrates, is used to promote significant tunability in the key MoS2 field-effect device parameters. This approach provides a powerful tool for modification of native substrate defect characteristics and allows for a wide range of property modulations. Our results signify the role of intrinsic and extrinsic defects in the physical properties of MoS2 and unveil strategies that can utilize these characteristics.