The fungal pathogen Diplocarpon rosae causes rose blackspot disease, a serious problem for roses (Rosa) in the managed landscape. To prevent this disease, homeowners and professional growers often apply chemical fungicide. However, increased use of fungicides poses an environmental hazard and an economic burden to the user. New landscape rose cultivars like ‘Knockout’ possess increased disease resistance, but the biological basis for this resistance is still unknown. To investigate the potential role of leaf cuticle in blackspot resistance in rose, five rose cultivars known to vary greatly in blackspot resistance were examined for variation in the major lipids of the leaf cuticle, specifically the monomers of the cutin polyester and the free cuticular waxes. This is the first report of cutin monomers in the Rosa genera. The rose cultivars selected for this study were ‘Knockout’, ‘Mister Lincoln’, ‘Garden Party’, ‘Purple Passion’, and ‘Bicolor’. ‘Knockout’ and ‘Garden Party’ had significantly lower total cutin monomer amount per leaf area than the other cultivars, whereas the most cutin monomers were observed on ‘Purple Passion’, ‘Bicolor’, and ‘Mister Lincoln’. Five major cutin monomers (mostly hydroxylated 16 carbon fatty acids) dominated the cutin profiles of both adaxial and adaxial surfaces of all cultivars, with the 10,16-dihydroxy hexadecanoic acids being most abundant. The proportion of 10,16-dihydroxy hexadecanoic acids was slightly higher in the adaxial than abaxial leaf cuticles of all cultivars. Correspondingly, other cutin monomers were relatively lower in the adaxial cuticle, except 16-hydroxy hexadecanoic acid that differed little. Uniquely, this is the first report of cutin monomer composition of isolated abaxial and adaxial leaf cuticles of any plant. Total leaf cuticular wax amounts were lowest on ‘Purple Passion’ and ‘Knockout’, intermediate in ‘Mister Lincoln’ and ‘Garden Party’, and highest on ‘Bicolor’, with alkanes as the most abundant wax class. Consistent with previously published disease susceptibility ratings, our visual scores showed that ‘Knockout’ was most resistant to blackspot pathogen infection with a visual disease rating score of 1.0, followed by ‘Mister Lincoln’ at 1.8, ‘Garden Party’ at 5.4, ‘Bicolor’ at 7.5, and ‘Purple Passion’ with the most visible disease damage at 8.8. Regression analysis revealed that the alkane and ester proportions were most closely associated with blackspot disease susceptibility ratings, being inversely (R2 = 0.63, P = 0.05) and directly (R2 = 0.81, P = 0.05) correlated, respectively. More studies on the role of cuticle in rose susceptibility to blackspot are now clearly warranted.