BackgroundOilseed rape (Brassica napusL.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungusSclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance inB. napusis quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding inB. napus.ResultsHere, we performed a genome-wide association study (GWAS) using a natural population ofB. napusconsisting of 222 accessions to identifyBnaA08g25340D(BnMLO2_2) as a candidate gene that regulates the SSR resistance.BnMLO2_2was a member of seven homolog genes ofArabidopsis Mildew Locus O 2(MLO2) and the significantly SNPs were mainly distributed in the promoter ofBnMLO2_2, suggesting a role ofBnMLO2_2expression level in the regulation of SSR resistance. We expressedBnMLO2_2in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues ofB. napusrevealed thatBnMLO2_2had the most expression level in leaf and silique tissues among all the 7BnMLO2members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis,mlo2plants displayed reduced resistance to SSR, whereas overexpression ofMLO2conferred plants an enhanced SSR resistance. Moreover, a higher expression level ofMLO2showed a stronger SSR resistance in the transgenic plants. The regulation ofMLO2in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion ofMLOfamily inBrassicacrops.ConclusionOur study revealed an important role ofBnMLO2in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance inB. napusand also new insights into understanding ofMLOfamily evolution inBrassicacrops.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02325-z.