A new cyst nematode, herein described as Globodera agulhasensis n. sp., was found parasitising Senecio burchelli in the Western Cape Province, South Africa. Second-stage juveniles are characterised by a well developed stylet of 23.5 (22.5-24.8) μm with rounded to anteriorly flattened knobs. The dorsal pharyngeal gland outlet is 4.4 (3.5-6.5) μm posterior to the stylet knobs. The tail is 56 (49-64) μm long and the length of the hyaline region is 25 (19-29) μm. The cysts are characterised by their ovate to spherical shape, short neck, the presence of subcuticular punctations over the entire body and the absence of bullae or vulval bodies. Six to 12 cuticular ridges/lines are present on the outer surface of the cyst between the anus and vulval basin. Granek's ratio is 1.7 (1.0-3.0), the vulval basin diam. 17.6 (11.7-26.1) μm and the distance between vulval basin and anus is 28.6 (19.1-47.0) μm. Males have a stylet length of 26.1 (24.4-27.7) μm and spicule length of 30.3 (27.2-33.8) μm with a rounded tip. Females have a stylet length of 22.1 (19.0-24.4) μm, a large median bulb almost filling the body diam., and a short vulval slit 4.2 (3.2-6.6) μm long. Phylogenetic relationships of G. agulhasensis n. sp. with other species of the genus, inferred from ITS-rRNA sequences by using the neighbour-joining (NJ), maximum likelihood (ML) and maximum parsimony method (MP), indicate that G. agulhasensis n. sp. is included in the clade of Globodera sp. that parasitise non-solanaceous plants, forming a monophyletic group with unidentified Globodera spp. from Portugal, G. millefolii and G. artemisiae. For diagnostic purposes, three restriction enzymes, Hpy8I, RsaI and XceI were selected as being able to discriminate between G. agulhasensis n. sp. and other Globodera spp. present in South Africa.