Key message: Rose has 19 MLO genes. Of these, RhMLO1 and RhMLO2 were shown to be required for powdery mildew infection, which suggests their potential as susceptibility targets towards disease resistance. Abstract: Powdery mildew, caused by Podosphaera pannosa, is one of the most serious and widespread fungal diseases for roses, especially in greenhouse-grown cut roses. It has been shown that certain MLO genes are involved in powdery mildew susceptibility and that loss of function in these genes in various crops leads to broad-spectrum, long-lasting resistance against this fungal disease. For this reason, these MLO genes are called susceptibility genes. We carried out a genome-wide identification of the MLO gene family in the Rosa chinensis genome, and screened for allelic variants among 22 accessions from seven different Rosa species using re-sequencing and transcriptome data. We identified 19 MLO genes in rose, of which four are candidate genes for functional homologs in clade V, which is the clade containing all dicot MLO susceptibility genes. We detected a total of 198 different allelic variants in the set of Rosa species and accessions, corresponding to 5–15 different alleles for each of the genes. Some diploid Rosa species shared alleles with tetraploid rose cultivars, consistent with the notion that diploid species have contributed to the formation of tetraploid roses. Among the four RhMLO genes in clade V, we demonstrated using expression study, virus-induced gene silencing as well as transient RNAi silencing that two of them, RhMLO1 and RhMLO2, are required for infection by P. pannosa and suggest their potential as susceptibility targets for powdery mildew resistance breeding in rose.