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Ecological restoration is predicated on our abilities to discern plant taxa. Taxonomic identification is a first step in ensuring that plants are appropriately adapted to the site. An example of the need to identify taxonomic differences comes from big sagebrush (Artemisia tridentata). This species is composed of three predominant subspecies occupying distinct environmental niches, but overlap and hybridization are common in ecotones. Restoration of A. tridentata largely occurs using wildland collected seed, but there is uncertainty in the identification of subspecies or mix of subspecies from seed collections. Laboratory techniques that can determine subspecies composition would be desirable to ensure that subspecies match the restoration site environment. In this study, we use spectrophotometry to quantify chemical differences in the water-soluble compound, coumarin. Ultraviolet (UV) absorbance of A. tridentata subsp. vaseyana showed distinct differences among A.t. tridentata and wyomingensis. No UV absorbance differences were detected between A.t. tridentata and wyomingensis. Analyses of samples from > 600 plants growing in two common gardens showed that UV absorbance was unaffected by environment. Moreover, plant tissues (leaves and seed chaff) explained only a small amount of the variance. UV fluorescence of water-eluted plant tissue has been used for many years to indicate A.t. vaseyana; however, interpretation has been subjective. Use of spectrophotometry to acquire UV absorbance provides empirical results that can be used in seed testing laboratories using the seed chaff present with the seed to certify A. tridentata subspecies composition. On the basis of our methods, UV absorbance values < 2.7 would indicate A.t. vaseyana and values > 3.1 would indicate either A.t. tridentata or wyomingensis. UV absorbance values between 2.7 and 3.1 would indicate a mixture of A.t. vaseyana and the other two subspecies.