In Ontario, Canada, beta-thalassemia is easily detected through measurement of hemoglobin A2, but most laboratories do not do exhaustive DNA investigations for alpha-thalassemia. Therefore, the prevalence of thalassemia in microcytic samples for hemoglobinopathy investigation in Ontario is unknown. To address this, we performed a prospective cohort study in which samples referred for hemoglobinopathy investigation were also evaluated for alpha-thalassemia by DNA testing. Of 800 samples submitted, 664 were evaluable. Of the 664 patients represented, 163 (24.5%) were beta-thalassemia major carriers, 68 (10.2%) were hemoglobin Bart's hydrops fetalis carriers and, in total, 361 (54.4%) had some form of thalassemia. We conclude that microcytosis due to thalassemia is common in Ontario and that major forms of thalassemia, including forms predisposing to hemoglobin Bart's hydrops fetalis and beta-thalassemia major, are frequent. This illustrates the importance of adequate prenatal and laboratory investigation for these abnormalities in Ontario and other similar multiethnic jurisdictions worldwide.