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Hand separation of roots of 2 or more plants species from soil cores is a tedious and labor-intensive task. Our objective was to determine whether near-infrared reflectance spectroscopy (NIRS) could be employed to estimate root biomass proportions in binary mixtures of alfalfa (Medicago sativa L.) with each of 4 grasses. Grasses chosen for experimentation were crested wheatgrass (Agropyron cristatum L.), intermediate wheatgrass [Thinopyrum intermedium (Host) Barkworth & D.R. Dewey], an intergeneric hybrid [Elytrigia repens (L.) Nevski × Pseudoroegneria spicata (Pursh) Love], and Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski]. In the first experiment, roots from single-species field plots were washed from soil cores, dried, ground, and mechanically mixed in preselected alfalfa-grass ratios in which the percentage of grass varied from 0 to 100. Equations to measure the proportion of alfalfa or grass were developed from near infrared reflectance data using 84 randomly selected samples. In the second experiment, the 5 plant species were grown in greenhouse pots in pure stands and in binary mixtures that included all combinations of the grasses. Root systems were separated while attached to the topgrowth, dried, and ground. Tissues from single species treatments were mixed and calibration equations developed from these mixtures were used to estimate the proportion of alfalfa and the proportion of grass in samples. Samples contained either one type of root or a mixture of roots in proportions similar to those that occurred naturally in the pots. Coefficients of determination (r2) between the estimated and the actual root mass ratios ranged from 0.92 to 0.99. Determination of the proportion of grass in the samples was more accurate and precise than determination of the proportion of alfalfa. After the appropriate calibration equations have been developed, NIRS is more efficient than hand separation for estimating alfalfa-grass root mass ratios. The utility of the techniques can be increased by developing equations that encompass more complex mixtures and a wider range of environmental circumstances. This material was digitized as part of a cooperative project between the Society for Range Management and the University of Arizona Libraries. The Journal of Range Management archives are made available by the Society for Range Management and the University of Arizona Libraries. Contact lbry-journals@email.arizona.edu for further information. Migrated from OJS platform August 2020