Toxic marine dinoflagellates from the genus Alexandrium have been responsible for paralytic shellfish poisoning (PSP) throughout the world. Their monitoring relies on spatial and temporal sampling strategies and requires the reliable identification and enumeration of vegetative stages in order to enable the development of early warning policies. The accurate discrimination between Alexandrium species is labour intensive and requires taxonomic expertise as the genus contains morphologically similar toxic and non-toxic species. In Ireland, PSP outbreaks so far have been limited to Cork Harbour, a retentive inlet located on the south coast of the country, where the causative organism has been identified as A. minutum. Recently, the non-toxic and morphologically similar species A. andersoni has been detected on the south west coast of Ireland. In routine monitoring, Alexandrium spp. are identified on the basis of morphological features by conventional light microscopy, a method which does not allow their characterization at the species level. The development and application of large subunit (LSU) rRNA-targeted oligonucleotide probes for the detection and quantification of A. minutum (Global clade) and A. andersoni by whole-cell fluorescent in situ hybridization (FISH) is reported. The specificity and sensitivity of the two probe sets (MinA and AndA′+C) were evaluated against Alexandrium species, including A. tamarense, A. tamutum and A. ostenfeldi, and a range of common dinoflagellates usually co-occurring with Alexandrium in Irish coastal waters. No cross-reactivity was observed with any of the strains tested or with phytoplankton species present in field samples rich in dinoflagellates. The format of the assay overcame possible matrix effects, such as probe adsorption, and allowed the reliable labelling of at least 1000 cells. Furthermore, the simultaneous use of calcofluor during the assays permitted the confirmation of the probe diagnostics by examining the general plate structure of the thecae of labelled cells.