Blackberries (Rubus subgenus Rubus) are a major berry crop consumed globally for being a rich source of anthocyanins and antioxidants, and their unique flavor. However, breeding for fruit improvement in blackberry has been significantly hindered by the scarcity of genomic resources and the genetic complexity of traits. The blackberry genome has been particularly challenging to assemble, largely due to its polyploid nature. We present the first chromosome-scale and haplotype-phased genome assembly for the cultivated primocane-fruiting, thornless tetraploid blackberry selection BL1 (Rubus L. subgenus Rubus Watson). The tetraploid genome assembly was generated using the Oxford Nanopore Technology (ONT) and Hi-C scaffolding, comprising 919 Mb placed on 27 pseudochromosomes with an N50 of 35.73 Mb. The assembly covers >92% of the genome length and contains over 98% of complete BUSCOs. Repetitive sequences constitute 57% of the assembly, with the long terminal repeats (LTR) being the most abundant class. A total of 87,968 protein-coding genes were predicted, of which, 82% were functionally annotated. Gene expression analyses identified candidate genes and transcription factors related to thornlessness in blackberries, including MYB16, lysine histidine transporters, PDR1, Caffeoyl-CoA, glycosylphosphatidylinositol-anchored lipid protein transfer 1, DRN, beta-ketoacyl reductase, and homocysteine S-methyltransferase 3. The utility of this genome has been demonstrated in this study by identifying candidate genes related to thornlessness in blackberry. Resequencing of tetraploid blackberry cultivars/selections with different horticultural characteristics revealed genes that could impact fruiting habit and disease resistance/susceptibility. This tetraploid reference genome will serve as a valuable resource to accelerate genetic analysis and breeding of this important berry crop, enabling the development of improved varieties with enhanced traits.