<p>Hyperspectral imaging (HSI) captures both spatial and spectral information simultaneously, enabling accurate discrimination of targets that are difficult to distinguish using conventional color imaging techniques. As a result, HSI has been widely applied across various fields, including remote sensing, industrial inspection, and biomedical diagnostics. Although many HSI methods have been developed, their imaging specifications—such as spatial and spectral resolution and acquisition speed—are largely constrained by the optical components employed. These limitations pose significant challenges in dynamic imaging environments or when the target’s size and shape vary over time. In this study, we present a novel HSI technique utilizing a digital micromirror device (DMD) to enable high spectral resolution imaging with adjustable spectral acquisition regions. The DMD operates in a binary mode, reflecting light toward two discrete angles based on input patterns, thereby facilitating the simultaneous acquisition of spectral data and wide-field images. The spectral acquisition regions are clearly visualized as darkened areas in the wide-field image, eliminating the need for post-imaging registration. The proposed DMD-based HSI method demonstrates high fidelity in spectral data acquisition and enables spatially resolved spectral imaging. Additionally, we validate its applicability to biomedical applications by successfully differentiating spectral profiles of normal and cancerous tissues from a H&amp;E-stained slide. Collectively, the DMD-based HSI approach offers a versatile and practical imaging solution in cases requiring high spectral resolution under dynamic imaging conditions.</p>