Rose is one of the major ornamental plants. The selection of glabrous cultivars is an important breeding target but remains a difficult task due to our limited genetic knowledge. Our objective was to understand the genetic and molecular determinism of prickles. Using a segregating diploid rose F1 population, we detected two types of prickles (glandular and non-glandular) in the progeny. We scored the number of non-glandular prickles on the floral and main stems for three years. We performed QTL analysis and detected four prickle loci on LG1, 3, 4 and 6. We determined the credible interval on the reference genome. The QTL on LG3 is a major locus that controls the presence of prickles, and three QTLs (LG3, 4 and 1) may be responsible for prickle density. We further revealed that glabrous hybrids are caused by the combination of the two recessive alleles from both parents. In order to test whether rose prickles could originate from a ‘trichome-like structure,’ we used a candidate approach to characterize rose gene homologues known in Arabidopsis, involved in trichome initiation. Four of these homologues were located within the overlapping credible interval of the detected QTLs. Transcript accumulation analysis weakly supports the involvement of trichome homologous genes, in the molecular control of prickle initiation. Our studies provide strong evidence for a complex genetic determinism of stem prickle and could help to establish guidelines for glabrous rose breeding. New insights into the relationship between prickles and trichomes constitute valuable information for reverse genetic research on prickles.