A drop of seawater contains numerous microspatial niches at the scale relevant to microbial activities. Examples are abiotic niches such as detrital particles that show different sizes and organic contents, and biotic niches resulting from bacteria-phage and bacteria-phytoplankton interactions. A common practice to investigate the impact of microenvironments on bacterial evolution is to separate the microenvironments physically and compare the bacterial inhabitants from each. It remains poorly understood, however, which microenvironment primarily drives bacterioplankton evolution in the pelagic ocean. By applying a dilution cultivation approach to an undisturbed coastal water sample, we isolate a bacterial population affiliated with the globally dominant Roseobacter group. Although varying at just a few thousand nucleotide sites across the whole genomes, members of this clonal population are diverging into two genetically separated subspecies. Genes underlying speciation are not unique to subspecies but instead clustered at the shared regions that represent ~6% of the genomic DNA. They are primarily involved in vitamin synthesis, motility, oxidative defense, carbohydrate, and amino acid utilization, consistent with the known strategies that roseobacters take to interact with phytoplankton and particles. Physiological assays corroborate that one subspecies outcompetes the other in these traits. Our results indicate that the microenvironments in the pelagic ocean represented by phytoplankton and organic particles are likely important niches that drive the cryptic speciation of the Roseobacter population, though microhabitats contributed by other less abundant pelagic hosts cannot be ruled out.