In order to identify effects of the cross-resistance and the function of resistant genes in germplasm M196, this study determined the resistance effect of M196 by spraying SU, IMI, TP, PB, and SCT herbicides with different concentration gradients in culture room and field, respectively. Results showed that the cross-resistance of SU and SCT was the strongest, while IMI, TP and PB were lower. The similar cross resistance to various herbicides was detected in the transgenic positive plants with resistance genes by functional validation in transgenic Arabidopsis. The cross-resistance of ALS herbicides was caused by the increased expression of mutant target gene and the decreased sensitivity of mutant target enzyme to herbicides in the transgenic Arabidopsis. The study provides a theoretical basis for preventing and solving the potential problems about weed resistance and drift of resistance gene that may arise from the widespread promotion of resistant varieties