Human embryonic stem cells (hESCs) have typical Raman signatures, but specific factors that contribute to variations in these signatures have not been reported to date. Furthermore, variations due to the passaging that is necessary for hESC culture maintenance could potentially distort these signatures. It is therefore important to characterize the impact of these culture manipulations on the Raman spectra to gain a better understanding of the origins and nature of their variations. Here we report on the Raman microspectroscopy of hESCs samples from maintenance cultures, complemented with periodic acid Schiff (PAS, carbohydrates) and 4′-6-diamidino-2-phenlyindol (DAPI, nuclei) staining. The component predominantly responsible for variations between spectra was spectrally identified as glycogen. Variations in the Raman map of the 480 cm−1 glycogen marker band corresponded with those of a PAS stain of the same sample area. The 785-nm Raman microspectra of hESC cultures examined daily after passaging showed that the same nonrandom spectral variances occurred at all time points after passaging. The pattern of these variances was identified as being due to glycogen spectral components. Our results help validate the previously observed spectral signatures of hESCs and further delineate and characterize the variations that can be expected in these signatures under normal maintenance culture conditions, and aid distinguishing them from those corresponding to differentiation, thus providing a benchmark for future studies. Copyright © 2010 John Wiley & Sons, Ltd.