Abstract: Gracilaria textorii is a red alga that is known for its high agar content, which is influenced by environmental factors such as salinity. To investigate the putative key genes involved in the agar synthesis pathway of G. textorii, transcriptome sequencing was performed to analyze the differentially expressed genes (DEGs) in G. textorii cultivated under low (10), normal (30), and high (50) salinity conditions, and four DEGs within the agar synthesis pathway, including the glucose-6-phosphate isomerase (GPI), mannose-6-phosphate isomerase (MPI), UDP-galactose-4-epimerase (GALE), and GDP-D-mannose-3’,5’-epimerase (GME) genes, were identified. Concurrently, the agar content in G. textorii increased after 15 d of cultivation at salinities 10 and 50. To further elucidate the correlation between the agar synthesis pathway genes and agar accumulation, the GPI, MPI (MPI-1, MPI-2), GALE, and GME genes were cloned based on the transcriptome data. Subsequently, transcript levels of these four genes and agar accumulation were analyzed under five salinity conditions: 10, 20, 30 (control), 40, and 50. The results demonstrated a significantly positive correlation between the relative agar accumulation and differences in relative transcript levels of the four genes across the different salinity treatments. This suggests that these four genes may be key determinants of the agar content in G. textorii.