Much more attention has been paid to the actinobacterial community in soils or water columns of aquatic habitats. However, there are few studies on their composition and diversity in lake sediments. Here, we used denaturing gradient gel electrophoresis and clone libraries of partial 16S rRNA gene to study the spatial variations of actinobacterial communities across 4 seasons in the surface sediments of the shallow, subtropical Taihu Lake. Cluster analysis based on fingerprints showed clear spatiotemporal variations of actinobacterial communities and higher seasonal variation than spatial heterogeneity. Based on clone libraries, this pattern was supported by the principal coordinates analysis in the phylogenetic context and by detrended correspondence analysis on the operational taxonomic unit table. Additionally, phylogenetic analysis showed that the putative freshwater-specific actinobacterial lineages (e.g., acI) were also detected in the lake sediments, which suggests that these subclusters may also adapt to the sediment environments. Summarily, our results suggested that actinobacterial communities of the surface sediments were more affected by seasonal variation than spatial heterogeneity in the intrahabitat of Taihu Lake.