Prickle-free blackberry (Rubus subgenus Rubus) canes are strongly preferred by growers due to food and worker safety concerns and damage to fruit from mechanical injury by prickles. This project was conducted to identify the genetic region responsible for prickle-free canes derived from the recessive 'Merton Thornless' source in autotetraploid blackberry using a genome-wide association study, develop diagnostic KASP markers for prickle-free canes, and determine the effects of allele dosage at the prickle-free locus on prickle density in two biparental populations. The prickle locus was located on chromosome Ra04 from 30.48 to 36.04 Mb in an extensive LD block, with the peak SNP located at 33.64 Mb. Two diagnostic KASP markers were developed that correctly predicted the phenotype of 97% and 96% of 626 diverse fresh-market blackberry genotypes from multiple breeding programs, respectively. Allele dosage at the prickle-free locus had a significant impact on prickle density, with duplex prickly genotypes having significantly higher prickle density than simplex genotypes in both biparental populations studied. Five potential candidate genes with functional annotations related to epidermal, trichome, and/or prickle development were identified within the prickle-free locus, but no nonsynonymous polymorphism within these genes were identified.