The synaptonemal complex protein 3 (SCP3) is a fundamental structural component of the synaptonemal complex and serves as a well-established molecular marker for investigating germ cell development and meiotic progression in teleost fishes. In this study, we focused on the marbled flounder (Pseudopleuronectes yokohamae), a cold-temperature flatfish species endemic to the Yellow and Bohai Seas that possesses considerable aquaculture potential due to its favorable growth performance and market value. However, despite its economic significance, the molecular mechanisms governing gonad development in this species—particularly the precise regulation of meiosis initiation during sexual differentiation—remain insufficiently characterized. Therefore, elucidating the expression dynamics of meiosis-associated genes such as scp3 is crucial for delineating the critical period of sex differentiation and deciphering the regulatory network underlying germ cell development in this commercially valuable flatfish. To address these knowledge gaps, this study had three main objectives. First, we aimed to clone and characterize the complete cDNA sequence of the scp3 gene from P. yokohamae. Second, we sought to examine its tissue distribution and stage-dependent expression patterns during gonadal development. Third, we planned to determine its precise cellular localization within gonadal tissues. These steps would help evaluate scp3 as a molecular marker for meiosis initiation and establish baseline data for understanding sex differentiation in this species. To achieve these goals, we collected specimens across various developmental stages. These included juveniles aged 60 to 150 days post-hatching (dph) and adults from 8 to 20 months post-hatch (mph). Total RNA was extracted from both gonadal and somatic tissues. Using integrated RT-PCR and RACE strategies, we obtained the full-length scp3 cDNA. We then performed comprehensive bioinformatics analyses to characterize the sequence architecture, predict protein structural features, and reconstruct phylogenetic relationships. Tissue distribution and developmental expression profiles were assessed using semi-quantitative RT-PCR and qRT-PCR. Cellular localization was determined by in situ hybridization (ISH) with digoxigenin-labeled RNA probes. Our results showed that the cloned scp3 cDNA is 1022 nucleotides long. It contains a 708-bp open reading frame encoding a 235-amino acid polypeptide. This protein includes characteristic structural motifs—a coiled-coil domain and a Cor1 conserved region—that are essential for synaptonemal complex assembly and function. Phylogenetic analyses revealed strong amino acid sequence conservation with other flatfish species, especially European flounder (Platichthys flesus) and plaice (Pleuronectes platessa). These species clustered within the Pleuronectiformes clade, confirming evolutionary conservation. Expression profiling indicated that scp3 transcripts are mainly restricted to gonads. Testicular expression levels were significantly higher than those in ovaries. Only minimal expression was detected in the brain, spleen, and heart. No expression was found in other somatic tissues examined. During juvenile development (60–150 dph), scp3 expression remained low until 130 dph. A statistically significant increase occurred at 140 dph, suggesting the initiation of meiosis around this time. In adult gonads, testicular scp3 expression rose progressively from 8 to 20 mph and consistently exceeded ovarian expression. Ovarian expression decreased initially from 8 to 16 mph, with a slight recovery at 20 mph. This pattern reflects fundamental differences in meiotic progression between spermatogenesis and oogenesis. Cellular localization via ISH confirmed cell-specific distribution. scp3 transcripts were mainly found in the cytoplasm of Stage II oocytes in ovarian tissue. In testicular sections, they were strongly associated with primary spermatocytes. Signal intensity was markedly higher in 20-month testis samples compared to 8-month ones, consistent with increased meiotic activity during testicular maturation. In conclusion, our study provides a comprehensive molecular characterization of the scp3 gene in P. yokohamae. The distinct spatiotemporal expression patterns support its validity as a reliable marker for meiosis initiation and progression. Key evidence includes upregulation at 140 dph, gonadal-predominant expression, testis-biased accumulation, and localization to meiotic cell types. These findings address important knowledge gaps in germ cell development regulation in this species. They also establish a foundation for future research on sex differentiation pathways. This work has important implications for developing monosex breeding approaches that utilize female growth advantages, thereby supporting sustainable aquaculture of P. yokohamae.