Alopecurus aequalis, a predominant weed species in wheat and oilseed rape fields, can no longer be controlled by mesosulfuron-methyl application after continuous use over several years. Based on dose–response studies, the putative resistant populations, JTJY-1 and JHHZ-1, were found to be resistant to mesosulfuron-methyl, with resistance index values of 5.5 and 14, respectively. Sensitivity assays of the mesosulfuron-methyl-resistant populations to other herbicides revealed that the JTJY-1 population had moderate or high cross resistance to sulfonylureas (SUs) and triazolopyrimidines (TPs), but displayed a low level resistance to imidazolinones (IMIs). JTJY-1 also had high multi-resistance to ACCase inhibitors, but remained susceptible to photosystem II inhibitors. The JHHZ-1 population was resistant to all ALS inhibitors tested, but was sensitive to ACCase inhibitors and photosystem II inhibitors. To clarify the molecular basis of resistance in JTJY-1 and JHHZ-1 population, the ALS and ACCase gene were sequenced. Two ALS mutations (Pro-197-Thr or Trp-574-Leu) were detected in the mesosulfuron-methyl-resistant plants. The ACCase gene analysis revealed that the resistant JTJY-1 population had an Ile-1781-Leu mutation. Furthermore, the presence of two different target site resistance (TSR) mechanisms (ALS and ACCase mutations) existing simultaneously in individual A. aequalis was firstly documented in the presented study.