AbstractBarnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is a noxious grass weed that infests rice fields and causes huge crop yield losses. In this study, we collected 12 E. crus-galli populations from rice fields of Ningxia Province in China and investigated the resistance levels to the acetolactate synthase (ALS) inhibitor penoxsulam and the acetyl-CoA carboxylase (ACCase) inhibitor cyhalofop-butyl. The results showed that eight populations exhibited resistance to penoxsulam and four populations evolved resistance to cyhalofop-butyl. Moreover, all four cyhalofop-butyl–resistant populations (NX3, NX4, NX6, and NX7) displayed multiple herbicide resistance to both penoxsulam and cyhalofop-butyl. The alternative herbicides bispyribac-sodium, metamifop, and fenoxaprop-p-ethyl cannot effectively control the multiple herbicide–resistant (MHR) plants. To characterize the molecular mechanisms of resistance, we amplified and sequenced the target site–encoding genes in resistant and susceptible populations. Partial sequences of three ALS genes and six ACCase genes were examined. A Trp-574-Leu mutation was detected in EcALS1 and EcALS3 in two high-level (65.84- and 59.30-fold) penoxsulam-resistant populations, NX2 and NX10, respectively. In addition, one copy (EcACC4) of ACCase genes encodes a truncated aberrant protein due to a frameshift mutation in E. crus-galli populations. None of the amino acid substitutions that are known to confer herbicide resistance were detected in ALS and ACCase genes of MHR populations. Our study reveals the wide spread of MHR E. crus-galli populations in Ningxia Province that exhibit resistance to several ALS and ACCase inhibitors. Non–target site based mechanisms are likely to be involved in E. crus-galli resistance to the herbicides, at least in four MHR populations.