We studied the petal number trait in a population obtained after self-pollination of R. x damascena f. trigintipetala following analysis of molecular markers which have previously been mapped near the major dominant locus Blfo/d6 determining this trait in other rose species including R. multiflora and R. hybrida. The results showed that the same genetic mechanism, which determines the petal number trait in R. multiflora and R. hybrida also controls the trait in R. x damascena f. trigintipetala and is related to the dominant effect of a single copy allele in the tetraploid genome of this species. We also analyzed the expression of several flower homeotic genes including R. x damascena APETALA1/FUL-like (paleo AP1 type), R. x damascena euAPETALA 3 (euAP3 line) and R. x damascena AGAMOUS in early stage flower buds corresponding to plants with double and simple flowers. The obtained results showed that only R. x damascena AGAMOUS was differentially expressed between the samples of double and simple flowers, its relative expression being upregulated 3.5-fold in simple flowers. We further cloned and sequenced the four genomic clones of R. x damascena AGAMOUS and studied the potential additive effect of this gene by analysing the segregation of its four alleles in the population of self-pollinated R. x damascena. Analysis of variance of the data for petal number and allele segregation did not show a statistically significant effect of any allele configuration of the AGAMOUS gene on the petal number trait in R. x damascena f. trigintipetala.