Maturity (degree of secondary wall development) and fineness (linear density) are important cotton quality and processing properties, but their direct measurement is often difficult and/or expensive to perform. An indirect but critical measurement of maturity and fineness is micronaire, which is one of the properties used to “class” or grade cotton. In the U.S., the Uster HVI is used to class or grade cotton fiber. The Cottonscope provides rapid, accurate, and precise measurements of cotton fiber maturity, fineness, ribbon width, and micronaire simultaneously in water using polarized light microscopy and image analysis. The Cottonscope results were compared to corresponding maturity, fineness, and micronaire results from the HVI and AFIS units. Cottonscope results, both direct and calculated, yielded very good agreement to corresponding HVI (micronaire, maturity) and AFIS (maturity, fineness) results. Cottonscope maturity distributions were superior to those from the AFIS unit, being more representative of expected shapes.