Micronaire is an important fiber quality parameter in the cotton and textile industry. Micronaire is a function of maturity (the degree of the fiber secondary wall development) and fineness (linear density). In prior research, bench-top near infrared (NIR) spectroscopy demonstrated the ability to measure micronaire, maturity, and fineness in and out of the laboratory. Small, portable handheld NIR instruments have been introduced and a program was established to measure micronaire in and outside the laboratory on seed cotton fiber and cotton lint, and consequently to measure maturity and fineness in the fiber. Adding new data to the original commercial lint-only samples, including data from different environments (laboratory and greenhouse) and fiber type conditions (laboratory ginned lint and seed cotton) made the calibration more robust, increasing the accuracy of the two NIR instruments (MicroNIR 2200 and Luminar5030) used in this experiment. Each instrument has its individual strengths. It is advisable to use the instrument that best fits the laboratory research objectives.