Copper foil in printed circuit board (PCB) transmission lines/interconnects is roughened to promote adhesion to dielectric substrates. It is important to characterize PCB substrate dielectrics and correctly separate dielectric and conductor losses, especially as data rates in high-speed digital designs increase. Herein, a differential method is proposed for separating conductor and dielectric losses in PCBs with rough conductors. This approach requires at least three transmission lines with identical, or at least as close as technologically possible, basic geometry parameters of signal trace, distance-to-ground planes, and dielectric properties, while the average peak-to-valley amplitude of surface roughness of the conductor would be different. The peak-to-valley amplitude of conductor roughness is determined from scanning electron microscopy images.