Due to the harmful nature of the rice blast fungus, Magnaporthe oryzae, it is beneficial to characterize field isolates to help aid in the deployment of resistance (R) genes in rice. In the present study, 252 field isolates of M. oryzae, collected from rice fields of Yunnan Province in China, were assessed for mating type, DNA fingerprinting, and disease reactions to differential rice lines. In total, 94 isolates (37.3%) were MAT1-1 and 158 (62.7%) were MAT1-2 based on polymerase chain reaction assays, and some of them were verified with the tester isolates. All MAT1-1 and MAT1-2 isolates were virulent to some of the International Rice Research Institute-Japan International Research Center for Agricultural Sciences monogenic lines harboring 22 major resistance genes as differential varieties. Three simple-sequence repeat markers were used to examine genetic diversity in all isolates. The existence of regional patterns of genetic diversity, sexual reproduction potential, and pathogenicity suggests that M. oryzae populations have been independently asexually adapted in rice fields during crop cultivation.