The nature and extent of reproductive isolation was examined between a new self-compatible hybrid species Senecio eboracensis (2n=40) and its parents, self-incompatible S. squalidus (2n=20) and self-compatible S. vulgaris (2n=40). The triploid F(1) of S. eboracensis x S. squalidus exhibited very low seed set (x=0.63%), and F(2) and F(3) progeny were able to recover nominal levels of fertility (x=23.9 and 9.7%), while F(1) and F(2) offspring of S. eboracensis x S. vulgaris showed reduced seed set (x=63.8 and 58.8%). In both cases, evidence from previous work indicates that reduced fertility is associated with meiotic chromosome mispairing, and is a likely consequence of recombining both parental genomes within this new taxon. No hybrid offspring between S. eboracensis and S. squalidus were found in the wild, and only one such hybrid was recorded among 769 progeny produced by S. eboracensis surrounded by S. squalidus on an experimental plot. Natural crossing between S. eboracensis and S. vulgaris was recorded to be very low (between 0 and 1.46%) in the wild, but rose to 18.3% when individuals of S. eboracensis were surrounded by plants of S. vulgaris. It was concluded that strong breeding barriers exist between the new hybrid species and its two parents. Prezygotic isolation between S. eboracensis and S. vulgaris is likely to be largely due to both species reproducing by predominant self-fertilisation. However, differences recorded for germination, seedling survival, time of flowering and characters associated with pollinator attraction, plus significant clumping of juvenile and adult conspecifics in the wild, probably also contribute to reproductive isolation and ecological differentiation.