Technical Abstract: Moisture is an important quality and processing property for the cotton industry. The standard reference method for determining the moisture content in cotton fiber is the ASTM oven method (gravimetric weight loss). Several concerns have expressed on its ability to measure the actual moisture content in cotton fiber. To address these concerns, a chemical method (automated Karl Fischer Titration, KFT) was developed as an alternate reference method for cotton fiber moisture. The chemical and oven moisture methods for cotton are compared to each other and to several commercially available laboratory and at-line moisture measurement instruments. The overall agreement between the oven and KFT methods was very good. The KFT method yields lower moisture results compared to the oven method. Four diverse moisture measurement systems and instruments were compared to both the oven and KFT methods. Comparable method agreement between moisture methods were obtained with both reference methods, with the best technical results were obtained with the portable thermal gravimetric and NIR spectroscopy methods. The KFT method agreements between the various instruments were lower compared to those observed with the standard oven method due to the oven method being used for instrument calibration.