The results presented in this work show for the first time that an electric field used to macroscopically align polymer nanofibers can also align polymer chains parallel to the fiber axis. This important result indicates that anisotropic structural properties (mechanical, electrical, etc.) can be induced in polymer nanofibers during the electrospinning process. Such uniaxially oriented nanofibers exhibit a variety of potential applications in biomedicine, microelectronics, and optics. A simple technique of vertical electrospinning with an electric field induced, stationary collection was employed to obtain the molecular orientation in polymer nanofibers. This manuscript describes the orientation process via electrospinning and verifies this molecular orientation in the polymer nanofibers using three independent methods: polarized Fourier transform infrared spectroscopy, polarized Raman scattering, and X-ray diffraction.