Cleavers, an annual or winter annual broadleaf weed in the Rubiaceae family, has become troublesome in the wheat fields of the Huang-Huai-Hai region in China due to its herbicide resistance. In North America the common name of the plant is stickwilly; in China it known as cleavers. Four populations of cleavers (JS-15, SD-10, JS-22, and AH-20) were collected from wheat fields in Jiangsu, Shandong, and Anhui provinces, where the plant was not being controlled with applications of florasulam. The aims of this study were to identify the herbicide resistance patterns and investigate the mechanism underlying florasulam resistance. Whole-plant dose-response experiments revealed a notable variation in the degree of resistance exhibited by three specific populations toward florasulam, in comparison to the most sensitive population (S and AH-9), with the highest resistance index reaching 841.4. A gene-sequencing assay for acetolactate synthase (ALS) found that plants that were resistant to ALS from the JS-15, JS-22, and AH-20 populations had a Trp-574-Leu mutation, while no known ALS resistance mutations were discovered in SD-10 plants. In vitro ALS enzyme activity assays also indicated that the extractable ALS from JS-15, JS-22, and AH-20 plants was greatly resistant to florasulam relative to plants that are susceptible. Additionally, according to the resistance rating system, all resistant populations were susceptible to carfentrazone-ethyl + MCPA-sodium and bipyrazone + fluroxypyr-methyl. AH-20, JS-15, and JS-22 exhibited resistance to selected ALS, 4-hydroxyphenylpyruvate dioxygenase (HPPD), and photosystem II (PS II) complex inhibitors, demonstrating RR and RRR resistance profiles, whereas AH-9 displayed sensitivity to virtually all tested agents. The SD-10 population, on the other hand, exhibited RR and RRR resistance to HPPD and PS II inhibitors, and sensitivity to tribenuron-methyl. These findings indicate that a target site–based mechanism drives resistance to the ALS inhibitor florasulam in populations of cleavers, but nontarget site resistance may also have contributed to resistance, but this was not investigated. Other herbicides with different sites of action were tested and were active against cleavers.