Proactive nutrition screening is an effective public health strategy for identifying and targeting individuals who could benefit from making dietary improvements for primary and secondary prevention of disease. The Dietary Screening Tool (DST) was developed and validated to assess nutritional risk among rural older adults. The purpose of this study was to evaluate the utility and validity of the DST to identify nutritional risk in middle-aged adults. This cross-sectional study in middle-aged adults (45–64 year olds, n = 87) who reside in Appalachia, examined nutritional status using an online health survey, biochemical measures, anthropometry, and three representative 24-h dietary recalls. The Healthy Eating Index (HEI) was calculated to describe overall diet quality. Adults identified by the DST with a nutrition risk had lower HEI scores (50 vs. 64, p < 0.001) and were much more likely to also be considered at dietary risk by the HEI (OR 11.6; 3.2–42.6) when compared to those not at risk. Those at risk had higher energy-adjusted total fat, saturated fat, and added sugar intakes and lower intakes of dietary fiber, and several micronutrients than those classified as not at risk by the DST. Similarly, the at-risk group had significantly lower serum levels of α-carotene, β-carotene, cryptoxanthin, lutein, and zeaxanthin but did not differ in retinol or methylmalonic acid compared with those not at risk. The DST is a valid tool to identify middle-aged adults with nutritional risk.