Objective Dorsoventral (D-V) polarity dysregulation in pharyngeal pouches (PPs) is a fundamental pathogenic mechanism underlying human 22q11.2 microdeletion syndrome and diverse congenital craniofacial malformations. Current understanding of PP development is hindered by the lack of high-resolution in vivo visualization tools and subpopulation-specific transcriptomic data, leaving the spatial molecular heterogeneity and functional division of PP sectors poorly defined. This study aims to establish a high-precision live-tracking model to identify the distribution of PP D-V subpopulations and resolve their molecular coordinate system. Methods A zebrafish endogenous reporter line, Ki(pdgfαa-P2A-EGFP), was generated via CRISPR/Cas9-mediated intron-targeted knock-in and crossed with the ventral PP marker line Tg(nkx2.3:mCherry) to create a dual-fluorescence tracing system. Confocal microscopy was employed to characterize the 3-D topological structure of PPs at 36 hours post-fertilization (hpf). PP tissues were microdissected, and three distinct subpopulations—dorsal (pdgfαa+), ventral (nkx2.3+), and intermediate transition cells (pdgfαa+/nkx2.3+)—were isolated by fluorescence-activated cell sorting (FACS). High-depth transcriptomic profiles were generated using Smart-seq2, followed by Principal Component Analysis (PCA), hierarchical clustering, and Gene Ontology (GO) enrichment analysis to dissect the molecular signatures of each subpopulation. Results We successfully generated a stable pdgfαa knock-in line with high transcriptional fidelity, enabling the precise identification of a three-compartment spatial architecture—dorsal, intermediate transition, and ventral zones—within the living zebrafish PP. PCA demonstrated distinct molecular identities for the three subpopulations, with a cumulative variance contribution of 96.7% from PC1 and PC2, confirming exceptional sample reproducibility and segregation. Functional analysis revealed that each subpopulation possesses a unique regulatory signature: the dorsal group is enriched in pathways related to neural tube development and neurotransmitter transport; the intermediate transition group is highly active in apoptosis, autophagy, and metabolic remodeling, suggesting its role as a "buffer zone" for boundary refinement; and the ventral group is predominantly specialized in muscle cytoskeleton assembly and calcium signaling, facilitating interactions with adjacent pharyngeal muscle precursors. Conclusion The zebrafish pharyngeal pouch is not a homogeneous structure but a highly organized signaling hub characterized by distinct D-V partitioning and functional heterogeneity. The identification of the intermediate transition zone as a site for fate remodeling and homeostatic maintenance provides a new conceptual framework for PP morphogenesis. The live-tracking model and the definitive transcriptomic landscape established in this study provide a precise molecular coordinate system for elucidating the origins of pharyngeal-derived organs and the developmental logic of related congenital defects.