We constructed a SSH (suppression subtractive hybridization) library based on two populations (Rs-C and Rs-P) of Radopholus similis from different host plants and exhibiting differences in pathogenicity on Musa paradisiaca and Anthurium andraeanum plants. In order to screen the clones with significant expression differences from the SSH library, a total of 2,400 clones was randomly selected and reverse northern blotting was performed on them. Out of the 2,400 clones, 89 clones showed significant expression differences. Out of sequencing these 89 clones, distinct sequences from 87 clones were obtained. Aligning the 87 distinct sequences against the non-redundant nucleotide database (nr) in NCBI, we found that five sequences were highly conserved with Rs-eng-1b. Two of five sequences with lengths of 467 base pairs (bp) (GW395922) and 742 bp (GW395923) were further employed to perform 5' RACE-PCR and 3' RACE-PCR, respectively. Subsequently, the complete length of Rs-eng-1b (EU414839) was obtained (1,427 bp). Our qPCR result showed that expression of Rs-eng-1b in the population Rs-C with high pathogenicity on host plants was approximately 2.7 times as much as the expression of Rs-eng-1b in the population Rs-P with low pathogenicity on host plants. Furthermore, the gene Rs-eng-1b from the Rs-C population also showed expression differences amongst four different development stages. The order of Rs-eng-1b relative expression abundance from high to low was females, juveniles, males, and eggs. We further used RNAi to test whether Rs-eng-1b of Rs-C population was responsible for pathogenicity which was the first RNAi work about Rs-eng-1b. The RNAi results showed that Rs-eng-1b expression had a positive correlation to pathogenicity of the population. The longer the RNAi treatment, the less pathogenic the nematode population was. Non-endogenous gfp dsRNA had no significant influence on the expression of Rs-eng-1b and pathogenicity of R. similis Rs-C population. In conclusion, all our evidence indicated Rs-eng-1b might be a crucial pathogenicity-related gene in R. similis.