ABSTRACT: Synechococcus cyanobacteria exhibits a highly diverse composition of photosynthetic pigments. The fluorescence excitation ratio (Ex495:545) of the blue light-absorbing photosynthetic pigment phycourobilin (PUB; λmax = 495 nm) to the green light-absorbing photosynthetic pigment phycoerythrobilin (PEB; λmax = 545 nm) differs between the pigment subtypes within Synechococcus Pigment Type 3, which is predominantly found in marine waters. In the present study, we developed a method to assess the Ex495:545 of natural Synechococcus subpopulations that were distinguished using a flow cytometer equipped with both blue (488 nm) and green (532 nm) lasers. A significantly positive linear relationship of Synechococcus strains was obtained between spectrofluorometry and dual-laser flow cytometry (y = 0.542x + 0.08, P < 0.05). Using this equation, the Ex495:545 of natural Synechococcus subpopulations were enumerated. An Ex495:545 below 0.60 only occurred in the coastal areas and co-occurred with other subpopulations with higher Ex495:545, ranging from 0.73 to 1.80. The co-occurrence of multiple Synechococcus subpopulations with different Ex495:545 was also observed along Japan’s coastline. However, in the open ocean, only one subpopulation was observed, with Ex495:545 above 1.63 at the surface and increasing by depth to 2.13. The application of this method to enumerate Ex495:545 will advance the study of pigment type diversity and the light acclimation strategy of Synechococcus in marine environments.