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The property of attenuation of light by cotton fiber is used for estimation of cotton fiber mass in cotton classification using high–volume instruments (HVIs). The objective of this study was to determine the light source types that have the best attenuation characteristics for the purpose of cotton fiber mass measurement leading to its use in HVI systems. The effect of different varieties and moisture content levels on the relationship between attenuation and cotton fiber mass was also studied. Attenuation values for different amounts of cotton were measured for three different cotton varieties at three different moisture content levels (low, medium, and high) by using an attenuation measurement system fabricated for this study. Seven types of light source (two ultraviolets, halogen, red light–emitting diode, two lasers, and near infrared) at different wavelengths against seven different cotton mass density levels were tested with three replications. The study showed that the relationship between cotton fiber mass density and the attenuation was nonlinear for all of the sources tested in the study. The effects of the variety and moisture content level were found to be insignificant only for the red LED and near infrared (NIR) sources. The NIR source has the widest attenuation range and was not affected by different varieties and moisture content levels. The cotton fiber mass density can be estimated from the percent attenuation of NIR using a single calibration equation (R 2 = 0.9709) for different varieties and moisture content levels with reasonable accuracy.