Spatial factors can play important and potentially confounding roles governing the presence, the absence, and the abundance of the diatom species and consequently influence the diatom-based bioassessment. To examine how spatial factors affect diatom community and biomonitoring, diatom community was sampled in three catchments with similar river length and watershed area across a large spatial scale along the Yangtze River, China. Redundancy analysis (RDA) showed that patterns of diatom distribution could be attributed not only to environmental variables (e.g., NO3-N, TN, WT, and COD), but also to spatial factors (e.g., elevation, latitude, and longitude). Partial RDAs partitioned the total variance of diatom data explained (64.7%) into three parts: pure spatial variables contributed the highest proportion (19.8%), followed by pure environmental variables (5.4%), and the shared effects by environmental and spatial sets explained a large proportion of diatom variance (39.5%). Recognizing the potential influences of spatial factors, further accumulations of diatom data within different regions would allow for the development of better diatom-based monitoring methods for watershed diagnosis and management.