Red raspberry (Rubus idaeus L.) production is an important high-value horticultural industry worldwide. The presence of prickles on the stems, petioles and undersides of the leaves complicates both field management and harvesting of raspberries. The utilization of cultivars with fewer prickles or prickle-free canes simplifies production. Morphological characteristics between prickled vs. prickle-free red raspberry, black raspberry (Rubus occidentalis L.), blackberry (Rubus hybrid), purple hybrid (R. occidentalis x R. idaeus), and hairy and prickled wineberry (Rubus phoenicolasius Maxim) were compared using scanning electron microscopy. While prickle development in all of the species followed a similar pattern, there were differences in the specific structures. In red raspberry and wineberry, prickles develop directly from glandular structures whereas in blackberry and black raspberry, prickles develop from the signal originated from glandular structures. Prickle development in purple hybrid was unique in the presence of one-sided lumps in the trichomes, which has not been seen in any other Rubus species. These evidences reinforce the association between presence of glandular trichomes and prickle development. RNA sequencing analysis was used to identify differentially expressed genes in the epidermal tissue of prickled ‘Caroline’ and prickle-free ‘Joan J’ and their segregating progeny. The differential expression analysis revealed significant downregulation of some vital development related transcription factors, including a MIXTA-like R2R3-MYB family member, MADS-box, AP2/ERF, NAC in prickle-free epidermis, which were confirmed by qRT-PCR. Genotype by sequencing combined with genome-wide association study was performed in a population segregating for prickles between ‘Joan J’ and ‘Caroline’ to identify the genomic region associated with prickle development in red raspberry. The study identified four SNPs on chromosome 4 associated with the phenotype and provided potential candidates from within ±100 kb of these associated SNPs. MYB16-like (MIXTA-like R2R3-MYB), MADS-box protein AGL30 and trichome birefringence-like 2 protein were among the top potential candidates for prickle production. CRISPR/Cas9 system was introduced to the nodal segment of prickled ‘Caroline’ by using Agrobacterium-mediated transformation. The potential candidate gene ‘MYB16-like (MIXTA-like R2R3-MYB family members)’ was targeted using two designed guide RNAs. Phenotype scoring and detection of mutation could not be performed because of failure to regenerate plantlets from the callus.