Water temperature fluctuations are considered to be a major factor affecting the immune functions and metabolic processes of scallops. To better understand the immune defense mechanisms of Yesso scallop, Patinopecten yessoensis following exposure to water temperature fluctuations, transcriptomic profiles in the gills from high-frequency fluctuations (HF_G), low-frequency fluctuations (LF_G), and no fluctuations (NF_G) groups were obtained using HiSeq™ 2500 (Illumina). For HF_G, scallops were transferred directly between 18 and 8 °C every 4 h and for 10 fluctuations, while scallops in LF_G were transferred between 18 and 13 °C every 12 h, for a total of 4 fluctuations. A total of 442,922,590 clean reads were generated in 9 libraries and then assembled into 210,780 unigenes with an average length of 705 bp and an N50 of 1253 bp. Based on sequence similarity, 54,529 unigenes (25.87%) were annotated in at least one database. Comparative analysis revealed that 696 unigenes differentially expressed in temperature stressed groups compared with the control, including 229 unigenes between HF_G and NF_G, and 548 unigenes between LF_G and NF_G, respectively. Additionally, among these differentially expressed genes (DEGs), there were 41 immune-related unigenes and 16 protein metabolism-related unigenes. These results provide fundamental information on the molecular defense mechanisms in the Yesso scallop gills after exposure to water temperature fluctuations.