Species within the toxic marine diatom genus Pseudo-nitzschia coexist in coastal and estuarine waters globally and are difficult to distinguish by microscopy. Here, we describe a sensitive, high throughput PCR-based Automated Ribosomal Intergenic Spacer Analysis (ARISA) approach to determine the relative abundance of Pseudo-nitzschia species within natural communities over space and time. The method was quantitatively validated using simplified mixtures of DNA or ITS1 standards from isolates of P. pungens, P. multiseries, and P. delicatissima. Relative abundance calculations based on ARISA profiles from these mixtures reflected input ratios, with minor deviations resulting from intraspecific variability. ARISA was used to identify and quantify at least eight species within Puget Sound and the eastern Strait of Juan de Fuca, Washington, USA: P. americana, P. australis/P. seriata, P. cuspidata, P. delicatissima, P. fraudulenta, P. fryxelliana, P. multiseries, and P. pungens; genotypes corresponding to P. pungens var. pungens and P. pungens var. cingulata were identified by environmental sequencing. The different species were significantly correlated with physical (temperature, salinity), biological (chlorophyll a fluorescence, oxygen), and/or chemical (ammonium, nutrient ratios) factors. The ability to determine shifts in the relative abundance of Pseudo-nitzschia species over spatial and temporal scales relevant to dispersion and selection facilitates dissection of the varied mechanisms driving vertical and horizontal species distribution patterns in hydrographically complex systems.