Root-knot nematodes (RKN) are important plant pathogens affecting rice in South-East Asia and southern Brazil in irrigated rice fields. In order to investigate the specific diversity of RKN associated with irrigated rice in southern Brazil, Meloidogyne spp. from Rio Grande do Sul (RS) and Santa Catarina (SC) States were characterised biochemically by esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Fifty-six Meloidogyne spp. populations were detected in 48% of rice samples, and a total of five esterase phenotypes were identified, four of which presented as drawn-out bands in different positions. In RS State, M. graminicola (Est VS1), Meloidogyne sp. 2 (Est R2) and Meloidogyne sp. 3 (Est R3) were identified, which corresponded to ca 80, 40 and 10% of samples, respectively. In SC State, M. graminicola, M. javanica (Est J3), Meloidogyne sp. 1 (Est R1), Meloidogyne sp. 2 and Meloidogyne sp. 3 accounted for ca 93.75, 12.50, 62.50, 12.25 and 6.25% of samples, respectively. The esterase phenotypes R1, R2 and R3 are new, never having been detected on rice before. Meloidogyne javanica showed a N1 Mdh phenotype (Rm: 1.0), while four other populations exhibited a N1a (Rm: 1.4) phenotype. All populations were tested with two SCAR markers specific to M. graminicola, which confirmed that, but no specificity was obtained with both markers in relation to the atypical populations analysed. Sequencing and phylogenetic analyses of internal transcribed spacer-rRNA (ITS) were performed to infer the phylogenetic relationship of these atypical Meloidogyne spp. populations. Meloidogyne sp. 1 grouped with the mitotic parthenogenetic species, while the two others (Meloidogyne sp. 2 and sp. 3) clustered with M. graminicola and other meiotic parthenogenetic species. Taken together, these data highlight the unprecedented specific diversity of RKN associated with irrigated rice in southern Brazil. Further morphological and phylogenetic studies involving these atypical isolates will be carried out to identify this complex of species.