Display omitted •Two major invasive Nymphaea mexicana hybrid groups are present in South Africa.•These hybrid groups likely share genetic material with Nymphaea odorata and Nymphaea alba.•Surveys for potential biocontrol agents can be expanded to include putative parents.•Understanding the genetic structure of invasives improves efforts to develop biocontrol. Hybridisation of alien invasive plants complicates efforts to develop biological control, because variations in the genetic makeup of the target plant can impact the survival of host specific agents that have evolved adaptations specific to the original host. To maximise the likelihood of success in a biological control program, potential agents should therefore be collected from populations in the region of origin that are genetically similar to plants in the invaded range. Molecular markers are useful tools to understand genetic contributions in hybrid populations, especially where morphological differentiation is difficult. Nymphaea mexicana Zuccarini (Nymphaeaceae) is an invasive alien plant in South Africa that is being targeted for biological control, but hybrids with intermediate morphological traits are also present at several sites. In this study, ISSR (inter simple sequence repeats) and ITS (internal transcribed spacer) markers were used to determine which Nymphaea species are likely to be putative parents of these hybrids, and morphological characters were also investigated to determine if genetic and morphological traits matched. Two major hybrid groups were identified, with one group clustering with Nymphaea odorata Aiton and the other clustering with Nymphaea alba L. A third, smaller group clustered with Nymphaea tetragona Georgi, whereas the remaining samples clustered with pure N. mexicana from the native range. Morphological features agreed with deductions drawn from molecular data. These results allow us to focus efforts to find compatible biological control agents and better understand the complicated genetic structure of N. mexicana and Nymphaea hybrids in South Africa.