Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is increasingly infesting imidazolinone-tolerant (IMI-T) rice (Oryza sativa L.) fields in China, and E. crus-galli imazamox resistance has become the major concern for weed management in IMI-T rice fields. In this study, the susceptible population JLGY-3 (S) and the suspected resistant population JHXY-2 (R) collected from IMI-T rice fields were used as research subjects. When treated with imazamox, the JHXY-2 (R) population showed a high level of herbicide resistance with a resistance index of 31.2. JHXY-2 (R) was cross-resistant to all five acetolactate synthase (ALS) inhibitors from different chemical families, but sensitive to herbicides inhibiting acetyl-CoA carboxylase. To understand the reason why JHXY-2 (R) was resistant to imazamox, we performed experiments to characterize potential target-site resistance (TSR) and non–target site resistance (NTSR) mechanisms. A Trp-574-Leu amino acid mutation in ALS and low imazamox ALS sensitivity were the main mechanisms underlying imazamox resistance in this JHXY-2 (R) population. There was no significant difference in ALS gene expression and ALS protein abundance between R and S populations. High-performance liquid chromatography–tandem mass spectrometry analysis showed enhanced metabolism of imazamox in JHXY-2 (R), which was in contrast to the results of pretreatment with a metabolic enzyme inhibitor. Treatments with cytochrome P450 monooxygenase/glutathione S-transferase (P450/GST) inhibitors did not alter the resistance level of JHXY-2 (R) against imazamox. Transcriptome sequencing showed that there was almost no significant difference in the expression of P450 and GST metabolic enzyme genes between R and S populations, and only GSTU1 showed a significant upregulation in the R population, further clarifying the NTSR mechanism of JHXY-2 (R). In conclusion, amino acid mutation and higher enzyme activity of ALS are the main causes of imazamox resistance in JHXY-2 (R). However, given the differences in imazamox residues in the leaves of E. crus-galli, there may still be undetectable NTSR mechanisms that are causing imazamox resistance in the R population.