Downy mildew, caused by Peronospora sparsa, is a systemic disease of blackberry that can cause significant production losses worldwide. P. sparsa is disseminated through asymptomatic infected blackberry plants. Currently, there is no clean plant certification program for blackberry downy mildew. The objective of this research was to determine the detection frequency of P. sparsa in blackberry roots, stems, and leaves for the development of sampling guidelines for asymptomatic nursery plants. Stems, emerging leaves, and fully expanded leaves collected from commercial ‘Natchez’, ‘Ouachita’, and ‘Caddo’ nursery stock were tested by nested PCR using published primers at the early bud sprouting, vegetative, and flowering to green berry phenological stages for the presence of P. sparsa. Initially, 90, 40, and 100% of Caddo, Natchez, and Ouachita, respectively, tested positive for P. sparsa. Detection of P. sparsa was inconsistent across cultivar, tissue type, plant, and phenological stage. Except for Natchez plants, P. sparsa was detected most frequently in leaves at all three phenological stages. Overall, detection of P. sparsa in Natchez was low, with the highest frequency of detection occurring at the vegetative stage. For Caddo and Ouachita, newly emerging leaves at the bud sprouting stage yielded a slightly higher frequency of detection than fully expanded leaves. The detection frequency in stem or root tissue ranged from 0 to 50% depending on the cultivar and phenological stage. In this study, we demonstrated that sampling newly emerging leaves at early bud break will result in an 80 to 90% detection frequency in cultivars that are susceptible to P. sparsa.