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Sucrose is the economic product from sugar beet. Disease resistance is often available in low-sucrose genotypes and, prior to the deployment of such novel genes as available into the cultivated spectrum, selection for increased sucrose content is required during introgression. The objective of this work was to evaluate a relatively rapid and inexpensive enzymatic-fluorometric microtiter plate assay for sucrose quantification in sugar beet root dry matter, both for progeny testing in the greenhouse and for evaluation of field-grown mother roots. As determined using HPLC, sucrose content in diverse populations of sugar and table beet assayed over various developmental stages ranged from 0.213 to 2.416 mmol g(-1) of dry matter, and these values were used as references for both refractometry and enzymatic-fluorometric assay. As expected, refractometric analysis generally overestimated sucrose content. Enzymatic-fluorometric analyses were reasonably well correlated with HPLC results for young greenhouse-grown root tissues (R2 = 0.976), and less so with older field-grown roots (R2 = 0.605), for unknown reasons. Enzymatic-fluorometric assays may be best deployed for progeny testing of young seedlings.