We have developed a method to use fast Fourier transformation (FFT) and inverse fast Fourier transformation (IFFT) to investigate hidden periodic structures on SEM images. We focused on samples of natural, play-of-color opals that diffract visible light and hence are periodically structured. Conventional sample preparation by hydrofluoric acid etch was not used; untreated, freshly broken surfaces were examined at low magnification relative to the expected period of the structural features, and, the SEM was adjusted to get a very high number of pixels in the images. These SEM images were treated by software to calculate autocorrelation, FFT, and IFFT. We present how we adjusted SEM acquisition parameters for best results. We first applied our procedure on an SEM image on which the structure was obvious. Then, we applied the same procedure on a sample that must contain a periodic structure because it diffracts visible light, but on which no structure was visible on the SEM image. In both cases, we obtained clearly periodic patterns that allowed measurements of structural parameters. We also investigated how the irregularly broken surface interfered with the periodic structure to produce additional periodicity. We tested the limits of our methodology with the help of simulated images. SCANNING 36:487–499, 2014. © 2014 Wiley Periodicals, Inc.