Climate changes interacting with human activities are raising the temperature in global oceans. To explore physiological responses of in situ phytoplankton assemblages to increasing temperatures, we conducted a shipboard experiment in tropical regions of the eastern Indian Ocean, Java Sea, and southern South China Sea. Throughout the surveyed areas, phytoplankton biomass (Chla) ranged from 0.09 to 0.86 μg L⁻¹ (median, 0.22 μg L⁻¹) in the surface and from 0.30 to 0.99 μg L⁻¹ (median, 0.50 μg L⁻¹) in maximal chlorophyll layer (DCM), respectively. Picophytoplankton that occupied 27–89% (79%) and 83–92% (88%) of total Chla in the surface and DCM layers, ranged from 0.32 × 10⁴ to 23.10 × 10⁴ cells mL⁻¹ (3.69 × 10⁴ cells mL⁻¹) and from 7.44 × 10⁴ to 25.70 × 10⁴ cells mL⁻¹ (12.60 × 10⁴ cells mL⁻¹), respectively. Synechococcus took up 30–97% (78%) of pico-cells compositions in the surface layer, while, in the DCM layer, Prochlorococcus took up 42–98% (91%). Moreover, the maximal photochemical quantum yield (F_V/F_M) of photosystem II (PS II) and the rapid light curve (RLC)-derived light utilization efficiency (α) were lower in the surface layer than that in the DCM layer, but the saturation irradiance (E_K) was higher. In particular, we found that acutely rising temperature decreased the F_V/F_M and α in both the surface and the DCM layers but increased the absorption cross-section (σ_PSII) of PSII photochemistry. Our results clearly indicate that the presently rising temperature adversely affects the photophysiology of natural phytoplankton assemblages in tropical oceans.