Skeletonema is a dominant genus of phytoplankton in China’s coastal waters and a pivotal primary producer in marine ecosystems. Multiple Skeletonema species are capable of forming harmful algal blooms (HABs), which impose severe adverse impacts on marine ecological environments. Time-series metabarcoding surveys have revealed pronounced thermal niche differentiation within the genus: Skeletonema marinoi dominates cold thermal niches, whereas Skeletonema tropicum favors warm habitats. Here, we integrated comparative genomics to dissect the divergent thermal adaptation strategies of these two species, which diverged approximately 12.6 million years ago (Mya). Our physiological assays demonstrated that the temperature supporting maximum growth rate (Tmax) of S. marinoi was 15 °C, with its upper thermal threshold for half-maximal growth (Tsup) at 30 °C and lower thermal threshold for half-maximal growth (Tsub) at 10 °C. For S. tropicum, the Tmax was 25 °C, with Tsup at 30 °C and Tsub at 15 °C. S. marinoi exhibited a substantially higher activation energy (Ea) and a greater maximum intrinsic growth rate relative to S. tropicum. Both species attained their maximum cell volumes at Tmax. Transcriptomic profiling uncovered congruent transcriptional regulatory patterns at Tsub and Tsup relative toTmax for S. marinoi, a conserved pattern also detected in S. tropicum. Nevertheless, the two congeners exhibited species-specific molecular regulatory machineries in response to thermal stress. Under suboptimal temperatures, S. marinoi displayed suppressed photosynthesis, ribosomal biogenesis and protein synthesis, alongside induced glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid metabolism, the ubiquitin–proteasome system, and DNA replication and repair pathways. In contrast, S. tropicum upregulated photosynthesis, ribosomal biogenesis and protein synthesis, while downregulating glycolysis, TCA cycle activity, the ubiquitin–proteasome system, and DNA replication and repair machinery. This investigation of two ecologically critical Skeletonema species lays a foundational framework for deciphering the molecular underpinnings of thermal adaptation in marine protists amid ongoing climate-driven ocean warming.