Pseudo-nitzschia is a cosmopolitan, planktonic diatom with some species known for the production of neurotoxin domoic acid associated with Amnesic Shellfish Poisoning events. Pseudo-nitzschia blooms have been regularly reported in the Shandong coastal regions (SCRs), yet the diversity of Pseudo-nitzschia species and its spatio-temporal dynamics remain poorly characterized. In this study, we employed metabarcoding analysis targeting the first internal transcript spacer (ITS1) of the ribosomal RNA gene (rDNA) to investigate species composition and the spatio-temporal dynamics across four seasonal research cruises, with the samples collected from May 2022 to February 2023. The ITS1-based metabarcoding analysis yielded 235 amplicao sequence variant (ASVs), confirmed 16 species/evolutionary clades, among which P. galaxiae emerged as the most dominant Pseudo-nitzschia species in SCRs, followed by P. multistriata that abundant in summer and autumn. The latter exhibited the highest intraspecific diversity (90 ASVs). The number of species recorded in this study far exceeded the previous morphology-based identification and 18S rDNA V4-based metabarcoding studies, demonstrating that ITS1 has higher species resolution for Pseudo-nitzschia in metabarcoding analysis. Seasonal succession and spatial heterogeneity were observed for all observed Pseudo-nitzschia species, with different Pseudo-nitzschia species including P. delicatissima and P. galaxiae showed different spatial preferences, reflecting their preferences to regional hydrodynamics (Yellow Sea Warm Current, coastal currents) and associated environmental gradients. In addition, co-occurrence of multiple clades within P. pungens and P. cuspidata complexes suggests sufficient niche breadth in the SCRs to support closely related lineages, implying potentially complex toxigenic risks. Furthermore, contrasting seasonal dynamics and environmental correlations were observed among different ASVs belong to a single Pseudo-nitzschia species (e.g., P. galaxiae and P. multistriata), demonstrating intraspecific diversity. Moreover, water temperature was identified as the primary driver of seasonal shifts, followed by nutrients (phosphate, silicate, nitrogen). Collectively, this study advances our understanding of Pseudo-nitzschia diversity in the SCRs by highlighting the importance of inter- and intraspecific diversity in shaping community responses to environmental variability.