Ultralow densities of crystalline nanospheres have been detected in amorphous polymer microspheres by utilizing the unique sensitivity of second-order nonlinear optical techniques to anisotropy. Vibrational sum frequency scattering (SFS) and X-ray diffraction (XRD) are used to quantify the crystallinity of amorphous poly(D,L-lactic acid) microspheres. While XRD does not display any crystallinity for the microspheres, SFS spectra and patterns are reminiscent of a heterogeneous microsphere that contains small crystalline domains. Nonlinear light scattering theory was used to model the data, and an average domain radius of 147 ± 65 nm was obtained. The degree of crystallinity (0.2%) was estimated by comparing XRD and SFS data obtained from the amorphous microspheres to similar data obtained from crystalline microspheres. We estimate a detection limit of 0.002% for SFS.