Sediment denitrification is the dominant nitrogen removal pathway in many aquatic habitats and can be regulated by local-, landscape-, and regional-scale factors. However, the mechanisms for how these multiple scale factors and their interactions affect the sediment denitrifying communities remain poorly understood. In this study, we investigated the community composition, diversity, and abundance of nitrite reductase genes (nirK and nirS)-encoding denitrifiers in 74 sediment samples from 22 Yangtze lakes using clone library and quantitative PCR techniques. Information of location, climate, catchment land use, water quality, sediment properties, and plant communities at each sampling site was analyzed to elucidate the effects of regional, landscape, and local factors on the characteristics of sediment denitrifying communities. Results of canonical correspondence analysis indicated that local factors were the key determinants of denitrifying community composition, accounting for over 20% of the total variation. Additionally, certain regional and landscape factors, including elevation and catchment built-up land, were also significantly related to the composition of denitrifying communities. Variance partitioning analyses revealed that diversity and abundance in the nirK denitrifier community were largely influenced by local factors, while those in the nirS community were controlled by both local and regional factors. Our findings highlight the importance of using different scale factors to explain adequately the composition and structure of denitrifying communities in aquatic environments.