The phytoplankton Phaeocystis globosa thrives in a wide range of marine regions and plays an important role in climate control. It can also form harmful algal blooms (HABs) that threaten environments and impact important coastal infrastructures. Mechanisms underlying the formation of P. globosa blooms still remain poorly understood. Accumulating evidence suggests that P. globosa has high genetic diversity and different P. globosa strains may have differential contributions to the development of P. globosa blooms. However, due to the lack of molecular markers with adequate resolution for distinguishing P. globosa genetic diversity, such differential contributions by different P. globosa strains could not be accurately ascertained. As such, high-resolution molecular markers need to be developed and applied to distinguish P. globosa genetic diversity. In this study, we undertook to define high-resolution molecular marker by assembling and comparing the whole chloroplast genomes of P. globosa strains isolated from different regions of the world. Through comparative analysis of P. globosa cpDNAs and detection of single nucleotide variations (SNVs), a molecular marker pgcp1 with improved resolution was developed. The pgcp1 demonstrated the highest resolution compared with other regions including 18S rDNA V4 region, 28S rDNA D1–D2 region and rbcL region, through genetic distance and phylogenetic analysis of 13 P. globosa strains. Molecular analysis of environmental samples and strains collected in multiple expeditions from a wide range of ocean regions including multiple regions in China, Vietnam, Thailand and Western Pacific using pgcp1 as the molecular marker displayed high genetic diversity of P. globosa with at least four major P. globosa clades. In conclusion, we have developed pgcp1 as a high-resolution molecular marker for the harmful algal bloom species P. globosa, which can be used to track intra-species genetic diversity and dynamics of P. globosa during bloom development.