Pseudo-nitzschia H. Peragallo is a marine diatom genus found worldwide in polar, temperate, subtropical and tropical waters. It includes toxigenic representatives that produce domoic acid (DA), a neurotoxin responsible for Amnesic Shellfish Poisoning. In this study we characterized two species of Pseudo-nitzschia collected from Port Stephens and the Hawkesbury River (south eastern Australia) previously unreported from Australian waters. Clonal isolates were sub-sampled for (i) light and transmission electron microscopy; (ii) DNA sequencing, based on the nuclear-encoded partial large subunit ribosomal RNA gene and internal transcribed spacer (ITS)-ITS1, 5.8S and ITS2 rDNA regions and, (iii) DA production as measured by liquid chromatography-mass spectrometry. Morphological and molecular data unambiguously revealed the species to be Pseudo-nitzschia micropora Priisholm, Moestrup & Lundholm (Port Stephens) and Pseudo-nitzschia hasleana Lundholm (Hawkesbury River). This is the first report of the occurrence of these species from the Southern Hemisphere and the first report of P. micropora in warm-temperate waters. Cultures of P. micropora, tested for DA production for the first time, proved to be non-toxic. Similarly, no detectable toxin concentrations were observed for P. hasleana. Species resolution and knowledge on the toxicity of local Pseudo-nitzschia species has important implications for harmful algal bloom monitoring and management.