The pathogenicity of 331 blast isolates (Pyricularia oryzae Cavara) collected from different regions and ecosystems for rice cultivation in Bangladesh was evaluated by compatibility on 23 differential varieties (DV), each harboring a single blast resistance gene, and susceptible ‘Lijiangxintuanheigu’ (LTH). A wide variation in virulence was found among the isolates, and 267 races were classified using a new designation system. Virulence of blast isolates against DV carrying the resistance genes Pia, Pib, Pit, Pik-s, Piz-t, Pi12(t), Pi19(t), and Pi20(t), as well as avirulence against those carrying Pish, Pi9, Pita-2, and Pita, was distributed widely in Bangladesh. Cluster analysis of the compatibility data on the DV initially classified the isolates into groups I and II. The virulence spectra of the two groups differed mainly according to the reactions of the DV to Pii, Pi3, Pi5(t), Pik-m, Pi1, Pik-h, Pik, Pik-p, and Pi7(t). Group I isolates were distributed mainly in rainfed lowlands, whereas group II isolates were found mainly in irrigated lowlands; however, there were no critical differences in geographic distribution of the blast isolates. In total, 26 isolates, which could be used to identify the 23 resistance genes of the DV on the basis of their reaction patterns, were selected as a set of standard differential blast isolates. To our knowledge, this is the first clear demonstration of the diversity and differentiation of blast races in Bangladesh. This information will be used to develop a durable blast protection system in that country.