This study evaluated the effect of flight altitude and canopy separation of container-grown Fire Chief™ arborvitae (Thuja occidentalis L.) on counting accuracy. Images were taken at 6, 12, and 22 m above the ground using unmanned aircraft systems. Plants were spaced to achieve three canopy separation treatments: 5 cm between canopy edges, canopy edges touching, and 5 cm of canopy edge overlap. Plants were placed on two different ground covers: black fabric and gravel. A counting algorithm was trained using Feature Analyst®. Total counting error, false positives, and unidentified plants were reported for images analyzed. In general, total counting error was smaller when plants were fully separated. The effect of ground cover on counting accuracy varied with the counting algorithm. Total counting error for plants placed on gravel (−8) was larger than for those on a black fabric (−2), however, false positive counts were similar for black fabric (6) and gravel (6). Nevertheless, output images of plants placed on gravel did not show a negative effect due to the ground cover but was impacted by differences in image spatial resolution.