The naturally occurring structure of articular cartilage has proven to be an effective means for the facilitation of motion and load support in equine and other animal joints. For this reason, cartilage has been extensively studied for many years. Although the roughness of cartilage has been determined from atomic force microscopy (AFM) and other methods in multiple studies, a comparison of roughness to joint function has not be completed. It is hypothesized that various joint types with different motions and regimes of lubrication have altered demands on the articular surface that may affect cartilage surface properties. Micro- and nanoscale stylus profilometry was performed on the carpal cartilage harvested from 16 equine forelimbs. Eighty cartilage surface samples taken from three different functioning joint types (radiocarpal, midcarpal, and carpometacarpal) were measured by a Veeco Dektak 150 Stylus Surface Profilometer. The average surface roughness measurements were statistically different for each joint. This indicates that the structure of cartilage is adapted to, or worn by, its operating environment. Knowledge of cartilage micro- and nanoscale roughness will assist the future development and design of treatments for intra- articular substances or surfaces to preserve joint integrity and reduce limitations or loss of joint performance.