In recent years, Galium aparine L. has not been controlled by tribenuron-methyl in major Chinese winter wheat fields. The objective of this study is to understand the molecular basis of the resistance mechanism to tribenuron-methyl in G. aparine and to find the specific mutation sites in amino acid sequence of acetolactate synthase (ALS) in the resistant biotype of G. aparine. Fragments that encode the ALS were amplified and cloned from susceptible (S) and resistant (R) biotypes of G. aparine to tribenuron-methyl and sequenced subsequently. The result showed that the nucleotide sequence of R-biotype of G. aparine differed from that of the S biotype with three amino acid substitutions, of which, the amino acid substitution of Trp574 (TGG) to Gly (GGG) is located in the highly conserved region Domain B. The substitution of Trp574 might be responsible for the resistance to tribenuron-methyl in the R-biotype of G. aparine. In recent years, Galium aparine L. has not been controlled by tribenuron-methyl in major Chinese winter wheat fields. The objective of this study is to understand the molecular basis of the resistance mechanism to tribenuron-methyl in G. aparine and to find the specific mutation sites in amino acid sequence of acetolactate synthase (ALS) in the resistant biotype of G. aparine. Fragments that encode the ALS were amplified and cloned from susceptible (S) and resistant (R) biotypes of G. aparine to tribenuron-methyl and sequenced subsequently. The result showed that the nucleotide sequence of R-biotype of G. aparine differed from that of the S biotype with three amino acid substitutions, of which, the amino acid substitution of Trp574 (TGG) to Gly (GGG) is located in the highly conserved region Domain B. The substitution of Trp574 might be responsible for the resistance to tribenuron-methyl in the R-biotype of G. aparine.