Species of Cylindrocladiella occur on a variety of hosts, where they are known to act as pathogens or saprobes. Eight species from this genus are currently recognised, of which five have been reported previously in South Africa: C. camelliae, C. elegans, C. lageniformis, C. parva and C. peruviana. Isolates of Cylindrocladiella were obtained from a newly established 99-Richter grapevine mother vine block exhibiting decline symptoms. The DNA phylogeny of these and additional isolates, also obtained from declining grapevines, was determined by sequencing the ITS (ITS 1, ITS2 and 5.8S) as well as (β-tubulin and histone H3 gene regions. This identified four species of Cylindrocladiella on grapevines in South Africa: C. lageniformis, C. parva, C. peruviana and a new species described here as C. viticola, which forms part of the C. infestans species complex. Pathogenicity trials, using stem inoculations on green and 1-year-old 99-Richter cuttings to determine the pathogenicity of selected isolates of these species, gave inconclusive results.