Three genetic sub-populations (clade I, II and III) of Pseudo-nitzschia pungens, the potential toxic marine diatom, are known to have distinguishable growth characteristics under different culture conditions and distinct distributed patterns in the world. However, to date their exact eco-physiological traits are unrevealed in fields due to lack of the method to detect and/or measure abundances of each sub-populations, hence, the qPCR (quantitative real-time polymerase chain reaction) assay was developed to detect and quantify the P. pungens cells of each clade. Designed two specific primer sets, Pcla12F/R (for clade I and II) and Pcla3F/R (for clade III) only could amplify each target genomic DNA. The, significant linear relationships (R2 > 0.998) was established between Ct (threshold cycle) value and the log of cell abundance for each clade. Through the melting curve analysis, comparisons for gene copy numbers among the three clades and spike test for field study, our qPCR assay was reliable to quantify the cell numbers of each clade. There was strong linear correlation (R2 > 0.990) between cell abundances as estimated by qPCR assay and direct counting via light microscope in spike test, and 0.24 (clade I), 0.25 (clade II) and 0.33 (clade III) P. pungens cells per mL were detected markedly upon the use of specific two-primer set. Finally, developed qPCR assay was applied on field samples successfully. Our study implicate that our qPCR assay is an accurate and sensitive technique to estimate the cell abundances of each clade of P. pungens in field works.