Preserved color markings in Paleozoic fossils are rare and have been hypothesized to reflect muscular attachment scars, diagenetic artifacts, or the altered remains of biochromes (organic pigments) or sclerochromes (structural colors) embedded in fossilized skeletal remains. More than 25 exceptionally well preserved phacopid trilobites with spotted patterns are described from the Middle Devonian of western and central New York State (USA). The small (∼0.23 mm) circular markings appear either brown on a lighter cuticle, or white on a darker cuticle. Thin section, scanning electron microscope imaging, energy-dispersive X-ray spectroscopy, and wavelength-dispersive spectroscopy elemental analyses show spots to be microcrystalline low-Mg calcite spheres embedded within the primary layer of the cuticle below the prismatic layer. Surrounding exoskeleton (low-Mg calcite) exhibits coarser crystallinity, typical lamellar structures, pore canals, and possible organic matrix, as expected for relatively unaltered trilobite skeletons. Potential diagenetic mineralogies or microstructures were not observed, making diagenesis an unlikely explanation. Spot distribution, morphology, and position in exoskeleton rule out a relationship with sites of musculature attachment and/or insertion. We suggest that spots represent original biologic structures manifested as either crystallographic or optical loci resulting in sclerochrome spots or possibly clear spots embedded in the cuticle that contrasted with a pigmented exoskeleton and may have served as windows to an underlying epidermis.