To identify the root-knot nematodes (RKNs) infecting onions in Yuanmou County, Yunnan Province, morphological and molecular biological techniques were used. Observation of their life cycle and pathogenicity was conducted through artificial inoculation experiments in a greenhouse. The results show that the morphological characteristics and measurement data of the second-stage juveniles (J2s) and females of RKNs infecting onion roots are highly consistent with those of Meloidogyne graminicola (M. graminicola). Sequence alignment of the mitochondrial DNA (mtDNA) COXI region and 28S rDNA D2-D3 region revealed sequence similarities of 99.51–100.00% and 99.48–99.61%, respectively, compared with M. graminicola sequences from GenBank. The specific primers Mg-F3/Mg-R2 reliably amplified a characteristic 369 bp band. Inoculation experiments confirmed that the pathogen can complete its entire life cycle (approximately 26 days (ds)) on the roots of healthy onion seedlings, inducing typical root-knot symptoms and females. In conclusion, the pathogen was identified as M. graminicola, which is the first report of M. graminicola infecting onions in China. This study provides important theoretical support for the molecular diagnosis of onion root-knot nematode disease and the green control of Allium L. vegetables in China.