2D lateral and vertical heterostructures have been actively studied for fundamental interest and practical applications. Although aberration-corrected electron microscopy and scanning probe microscopy have been used to characterize a wide range of 2D heterostructures, the 3D local atomic structure and crystal defects at the heterostructure interface have thus far defied any direct experimental determination. Now, a collaborative team from UCLA, Harvard University, MIT and UC Irvine demonstrates a correlative experimental and first principles method to determine the 3D atomic positions and crystal defects in a MoS2-WSe2 heterojunction with picometer prevision and capture the localized vibrational properties at the epitaxial interface. They observe various crystal defects, including vacancies, substitutional defects, bond distortion and atomic-scale ripples, and quantitatively characterize the 3D atomic displacements and full strain tensor across the heterointerface. The experimentally measured 3D atomic coordinates, representing a metastable state of the  heterojunction, are used as direct input to first principles calculations to reveal new phonon modes localized at the heterointerface, which are corroborated by spatially resolved electron energy-loss spectroscopy. In contrast, the phonon dispersion derived from the minimum energy state of the  heterojunction is absent of the local interface phonon modes, indicating the importance of using experimental 3D atomic coordinates as direct input to better predict the properties of heterointerfaces. Looking forward, it is expected that the ability to couple the 3D atomic structures and crystal defects with the properties of heterostructure interfaces will transform materials design and engineering across different disciplines.