The metabolite profiling of fruits of the herb Ligustrum lucidum Ait collected during different months has been performed using ultra‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry (UPLC/QTOFMS) and multivariate statistical analysis techniques. The markers such as oleuropein acid, neonuezhenide, specnuezhenide, oleuropein and ligustrosidic acid accountable for such variations were identified through the loadings plot of principal component analysis (PCA), and the tentative identification of the markers is completed by comparing the mass spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae and MS/MS data with those of the known compounds published. Furthermore, one of the chemical markers, such as specnuezhenide, which is water‐soluble, biologically active and also the predominant compound in this crude drug, was quantified by ultra‐performance liquid chromatography coupled with a tunable UV detector (UPLC‐TUV). The developed UPLC method provides good linearity (r 2 = 0.9991), repeatability (RSD = 2.96%), intra‐ and inter‐day precisions (RSD = 0.21%, 0.96%), with accuracies of 99.18–100.26% and a recovery of specnuezhenide of 97.57%. The fruits of L. lucidum Ait collected from August to December were tested. The results clearly show that the fruits of L. lucidum Ait harvested in October have the highest yields of specnuezhenide. It is also noted that the variations of content of specnuezhenide obtained by both methods have a strong correlation. This suggests that the newly proposed strategy is a reliable and simple method for the rapid discrimination of subtle variations, within the same plant species or strains, due to different seasonal collection times. Copyright © 2011 John Wiley & Sons, Ltd.