A new reference method for water content in lint cotton, based on Karl Fischer Titration, is highly selective for water and gives precise results. The aim of this study was to evaluate the influence of cultivar, defoliation time and gin-drying temperature on water content of lint cotton, raw and cleaned. Five cultivars were grown in the field in Mississippi in 2009 and subjected to two possible defoliation dates and gin-drying temperatures. The ginned lint underwent further processing including mechanical cleaning, and scouring and bleaching. Also, prediction models were developed to help understand how defoliation timing influences water content changes within cultivars. Overall, the mean water content across all samples analyzed was (%): raw, 7.83; cleaned, 7.69; and scoured and bleached, 8.10. Withincultivar range of the averaged water data from the various processing treatments was (%): raw, 0.19; mechanically cleaned, 0.13; and scoured and bleached, 0.08. The hypothesis that the different genetic backgrounds of the cultivars, defoliation dates and gin-drying temperatures would not affect water content of cleaned samples within cultivars was accepted as true, but only for the scoured and bleached samples. Model predictions of the change in water content with defoliation timing agreed well with experimental data. The data demonstrates that a change in water content in the botanical trash in ginned lint (raw cotton) may be confused with that due to a change in maturity. It is recommended that the correlations between water content and maturity be limited to cleaned cottons.