For the first time the strain-induced metastable beta-form crystal structure, with the extended chains adopting a planar zigzag conformation, was discovered in the macroscopically aligned electrospun nanofibers of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHx) collected across the air gap on aluminum foil and on the tapered edge of a high-speed rotary disk. The presence of the) beta-form crystal structure in the fiber mats was confirmed by wide-angle X-ray diffraction (WAXD) and Fourier transform infrared spectroscopy (FTIR). In addition, selected area electron diffraction (SAED) and AFM-IR were utilized to investigate the morphological and structural details of individual electrospun nanofibers. The SAED results confirmed a significant influence of the collection method on the crystal structure as well as the orientation level of the molecular chains in the crystals. The AFM-IR spectra of the single nanofibers matched well with the traditional FTIR spectra, but the finer features in the AFM-IR spectra were more distinct and better resolved. Based on the experimental results, new mechanisms for the generation of the beta-form crystalline structure in electrospun PHBHx nanofibers are proposed.