The primary goal of thematic accuracy assessment is to measure the quality of land cover products and it has become an essential component in global or regional land cover mapping. However, there are many uncertainties introduced in the validation process which could propagate into the derived accuracy measures and therefore impact the decisions made with these maps. Choosing the appropriate reference data sample unit is one of the most important decisions in this process. The majority of researchers have used a single pixel as the assessment unit for thematic accuracy assessment, while others have claimed that a single pixel is not appropriate. The research reported here shows the results of a simulation analysis from the perspective of positional errors. Factors including landscape characteristics, the classification scheme, the spatial scale, and the labeling threshold were also examined. The thematic errors caused by positional errors were analyzed using the current level of geo-registration accuracy achieved by several global land cover mapping projects. The primary results demonstrate that using a single-pixel as an assessment unit introduces a significant amount of thematic error. In addition, the coarser the spatial scale, the greater the impact on positional errors as most pixels in the image become mixed. A classification scheme with more classes and a more heterogeneous landscape increased the positional effect. Using a higher labeling threshold decreased the positional impact but greatly increased the number of abandoned units in the sample. This research showed that remote sensing applications should not employ a single-pixel as an assessment unit in the thematic accuracy assessment.