The model diatom Thalassiosira pseudonana is believed to be a single species with a global distribution, but it has not been confirmed previously whether isolates from different environmental and geographic origins are genotypically and phenotypically identical. In the present study, a polyphasic approach was employed to characterize nine clonal isolates, plus an additional replicate of one of the isolates, of the diatom T. pseudonana from culture collections to investigate whether there was any cryptic speciation in the publicly available strains of this species. Morphological analysis using scanning electron microscopy concluded that the strains were indistinguishable. Furthermore, conventional DNA barcoding genes (SSU rDNA, ITS1 and ITS2 rDNA and rbcL), revealed no nucleotide variation among the strains tested. On employing a whole genome fingerprinting technique, Amplified Fragment Length Polymorphism (AFLP), three clusters were revealed, although the level of variation between the clusters was surprisingly low. These findings indicate a low level of diversity among these cultured T. pseudonana strains, despite their wide spatial and temporal distribution and the salinity range of their original habitats. Based on the limited number of available strains, this suggests that T. pseudonana is a highly conserved diatom that nevertheless has an ability to tolerate wide ranges of salinity and populate varied geographic locations.