Large-scale roll-to-roll (R2R) fabrication of vertically oriented nanostructures via directed self-assembly of cylindrical block copolymer (c-BCP) thin films is reported. Nearly 100% vertical orientation of cylinders in sub-100 nm c-BCP films under optimized processing via a dynamic sharp temperature gradient field termed Cold Zone Annealing-Sharp or ‘CZA-S’ is achieved, with successful scale-up on a prototype custom-built 70 ft × 1 ft R2R platform moving at 25 μm/s, with 9 consecutive CZA units. Static thermal annealing of identical films in a conventional vacuum oven fails to produce comparable results. As a potential for applications, we fabricate high-density silicon oxide nanodot arrays from the CZA-S annealed BCP thin film template.