Abstract: In order to study substance migration during yellow spotting on cigarette paper, sugars and humectants were used as markers to investigate their variations in spotted cigarette paper by gas chromatography-mass spectrometry (GC/MS) after derivatization. The micro-morphology of spotted cigarette paper was characterized via scanning electron microscope (SME). The results showed that:1) The limits of quantification (LOQs) for 8 sugars and 4 humectants ranged from 0.27 to 8.17 mg/kg with the recoveries of 80%-116% at three spiked levels. 2) Yellow spots appeared on the paper of test cigarettes when relative humidity was higher than 80%, and the contents of fructose and glucose in the spotted cigarette paper were significantly higher than those before spotting. 3) At 80% relative humidity, the sugar content in cigarette paper increased with the increase of storage duration, the whiteness of cigarette paper reduced, and yellow spots formed after 24 h. 4) Micro-morphological analysis indicated that crystals formed on the inner surface of the spotted cigarette paper and the surface of cut fillers contacted with the yellow spots, which suggested that a large quantity of sugars was transferred from the cut fillers to cigarette paper when the cigarette became damp in a high humidity environment, meanwhile some soluble color substances were also transferred to the cigarette paper.