The aim of the present study was to evaluate the clinical efficacy of the single-shot dual-energy subtraction technique for obtaining portal images. We prepared two storage phosphor plates for this study. A 1 mm thick tungsten sheet was placed between the two storage phosphor plates. A single use of the double-exposure technique provides two portal images simultaneously (i.e., a standard image and a low-contrast image), using the same patient position and with no additional radiation delivered to the patient. A bone-enhanced image is created by image subtraction between these two images. For evaluation of clinical efficacy, three treatment sites--the brain, lung, and pelvis--were imaged. Ten sets of images were obtained for each site, and five landmarks were selected for each treatment site. The visibility of each landmark and the ease of overall verification for the selected treatment sites were assessed separately for the standard and bone-enhanced images. Four observers consisting of one radiation oncologist and three radiation therapists participated in the present study. For most of the landmarks studied, the bone-enhanced images were significantly superior to the standard images. Regarding the ease of overall verification, the bone-enhanced images were significantly superior to the standard images at all sites. The p-values of mean rating for the brain, lung, and pelvis were 0.002, 0.012, and 0.003, respectively. The bone-enhanced images obtained using our technique increased the image quality in terms of bone visibility, and are considered useful for routine clinical practice.