Tissues serve as the functional units of multicellular organisms with intricate spatial organizational complexity. Next-generation sequencing (NGS)-based spatially resolved transcriptomics (SRT) delineates in situ gene expression heterogeneities, establishes high-fidelity associations between transcripts and spatial pixels by segmenting a tissue section into spatial pixels, and elucidates the pivotal roles of cellular spatial organization in biological processes and complex pathological mechanisms. The synergistic integration of nucleic acid barcoding and high-throughput sequencing technologies has rapidly advanced the development of these spatial modalities regarding throughput, resolution, cost-effectiveness, and sensitivity. In this review, we summarize the state-of-the-art sequencing-based spatial transcriptomics, with a primary emphasis on the methodologies. We first introduce two major categories of typical nucleic acid barcoding platforms (in situ barcoding-based and barcoding array-based) used for spatial localization and transcriptome profiling. Then, the burgeoning trend of spatial transcriptomics towards spatiotemporal transcriptomics, which integrates SRT with the temporal dimension to provide more holistic landscapes of gene expression networks, is discussed. We also highlight spatiotemporal transcriptomics based on metabolic RNA labeling that provides unprecedented resolution to resolve transcriptome-wide dynamics in space and time. The emerging applications of these technologies in providing mechanistic insights into complex pathological mechanisms are also discussed. Finally, the perspectives on current bottlenecks and future direction of spatiotemporal transcriptomics are provided.