The ability to characterize individual electrospun fibers is essential in order to understand and control this complex process. In this paper, we demonstrate that confocal Raman microscopy is a powerful method to quantify molecular orientation and structure at the individual fiber level using poly(ethylene terephthalate) as a model system. Highly reproducible polarized spectra with an excellent signal-to-noise ratio were measured in 1 min or less for fibers with a diameter as little as 500 nm. The orientation of smaller fibers can also be probed using a calibration procedure. Our results reveal a very broad distribution of molecular orientation and structure within the samples: some individual fibers are completely isotropic and amorphous while others present a < P-2 > orientation parameter as large as 0.75. The development of this large orientation is accompanied by a gauche-to-trans structural conversion into the mesomorphous phase. Even the most highly oriented fibers only present a very small degree of crystallinity.