The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.