Cotton fiber length and maturity, two critical fiber qualities, are commonly determined in the U.S. by Uster high volume instrument (HVI) and advanced fiber information system (AFIS). The main objectives of this investigation were to compare how HVI lengths agree with AFIS lengths and to examine whether the fiber length is linked with fiber maturity between the Universal HVI length calibration cotton standards and diverse upland lint samples. HVI micronaire (MIC) and AFIS fineness showed insignificant differences from HVI length calibration cotton standards to lint samples. Although there were strong and significant correlations between HVI upper-half mean length (UHML) and either AFIS UQL (w) or AFIS L5% (n), the relationship between UHML and L5% (n) was better suited than between UHML and UQL (w) in scrutinizing fiber lengths. Meanwhile, analysis revealed a moderate correlation between AFIS L5% (n) length and AFIS maturity ratio (MR), indicating the possibility of improving AFIS L5% (n) length by regulating fiber MR development. Further, AFIS MR values were positive and moderate correlated with algorithmic MIR values of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectra. The results suggested the feasibility of the ATR FT-IR method along with MIR analysis in estimating AFIS MR rapidly away from fiber testing laboratories.