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Background: Ragweed is an invasive, highly allergenic weed predicted to expand its habitat with warming global temperatures. Several Ambrosia species have been identified in South Africa for well over a century; however, its presence remained undetected by allergists and aerobiologists until the development of an extensive aerospora monitoring system across South African urban areas since 2019. This paper presents the inventory of preliminary investigation of the Ambrosia airborne pollen and the taxonomic identification of ragweed species. Methods: Burkard volumetric spore traps for collecting pollen samples are set up in 9 South African cities (Johannesburg, Cape Town, Pretoria, Kimberley, Durban, Potchefstroom, Ermelo, Bloemfontein, and Gqeberha). Light microscopic identification was combined with environmental DNA metabarcoding analysis to confirm the species level of airborne Ambrosia at selected monitoring stations. Ragweed sensitisation was examined in Cape Town between February 2019 and February 2024, using Allergy Xplorer (ALEX2) multicomponent allergen array. Results: Ambrosia pollen was detected in 5 aerobiological monitoring stations over the sampling period (Durban, Kimberley, Pretoria, Potchefstroom, Johannesburg). Periods of 4 consistent pollination years were observed in Kimberley (min: 1; max: 16 p.g/m3) and Durban (min: 26; max: 66 p.g/m3). In Pretoria, ragweed pollen was detected for 2 years (2020–2021; 2022–2023) with average total annuals (5-17 p.g/m3). A peak flowering period between March and April was observed in Potchefstroom, and several ragweed pollen peaks were present between the end of December and the beginning of May in Durban. The highest number of Ambrosia pollen grains was recorded in Potchefstroom, with 308 grains, and a maximum peak of 47 p.g/m3. eDNA metabarcoding confirmed the presence of Ambrosia artemisiifolia and A.trifida species. The overall prevalence of Ambrosia-sensitisation amongst 673 tests (age range 7–72 years) was 8.2% (55/673), with no significant difference in sensitisation patterns between age groups. Conclusion: Our study confirms the need to monitor the spread of ragweed, and an increasing awareness of Ambrosia as an allergen of concern in Southern Africa. Extension of aerobiological networks and testing for Ambrosia sensitisation across urban and rural sites will be required.