Pseudo-nitzschia is a group of widely distributed planktonic diatoms. Some species produce the neurotoxin domoic acid. Herein, two new Pseudo-nitzschia species were described from Chinese coastal waters and the South China Sea after combining morphological and molecular data, together with biogeographical traits. Pseudo-nitzschia punctionis sp. nov. was similar to Pseudo-nitzschia bipertita morphologically, but differed in poroid structure, which was undivided in P. punctionis but divided in two sectors in P. bipertita. This difference corresponded to the presence of two hemicompensatory base changes (HCBCs) in the secondary structure of internal transcribed spacer 2. Pseudo-nitzschia polymorpha sp. nov. was sister to Pseudo-nitzschia limii phylogenetically, but distinct by the various shapes of perforations on the valve and copula, which was supported by four HCBCs. In a metabarcoding analysis, multiple new ribotypes were identified within the two new species, and intraspecific genetic divergences were analyzed. Metabarcoding data revealed that P. punctionis had a broader temperature range (12.9–30.5 °C) than P. polymorpha (22.3–30.5 °C). Within the two new species, different traits were found among the amplicon sequence variants regarding temperature and biogeography, representing different microevolutionary directions under environmental selection. The two new species had different biogeographical traits when compared to their closely related species. Domoic acid was detected in strains of P. punctionis at a concentration of 13.5–17.7 fg/cell, but the toxin was not found in strains of P. polymorpha. A combination of characters based on laboratory strains and field metabarcoding data provided more data for delimiting Pseudo-nitzschia species and gave new insights into their diversity and biogeography.