Hyperspectral Image Dataset for Focus Analysis (HIDFA) is composed by different hyperspectral (HS) images, acquired varying the working distance from the objective lens to produce different blurriness levels (11). HS images were captured from commercial samples (Brunel microscopes Ltd, England, UK) which include rat histology, leaf structures, stems, blood smears, and freshwater algae. Captures were taken using 5×, 10× and 20× magnification lenses in a HS microscope previously employed for different histology applications.