The universal attenuated total reflectance Fourier transform infrared (UATR-FTIR) equipped with a ZnSe-diamond composite crystal accessory was used to investigate the implication of trehalulose in the formation of yarn defects during rotor spinning of moderately sticky cotton mixes. One cotton bale contaminated with aphid honeydew and one contaminated with whitefly honeydew were selected. Mixes having moderate levels of stickiness were prepared from these two bales by mixing sticky cotton with non-sticky cotton. These cotton mixes were processed and the yarn defects were collected and analyzed using UATR-FTIR. The results obtained showed a high correlation between the FTIR spectra of the yarn defects from the whitefly honeydew-contaminated mixes and the spectra of artificial reconstituted whitefly honeydew. No significant difference was seen between the spectra of yarn defects from aphid honeydew-contaminated cotton and those of non-contaminated cotton. The FTIR and Raman studies of the hydration process of trehalulose showed a drastic impact of moisture absorption on the intensities of the vibration modes of trehalulose. These results all indicate that trehalulose is the primary concern when forming yarns from cotton with moderate honeydew contamination.