The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a transboundary polyphagous insect pest which preferentially feeds on grain crops. The FAW was responsible for economic damage to maize crops in more than 44 African countries by 2019 accounting for the loss of over US$2–5.5 billion. The parallel cultivation of Bt-maize expressing Cry1Ab protein and indigenous maize varieties in some African countries was aimed to enhance output and mitigate the harmful impact of the Lepidopteran insect below the economic injury level. A total of six maize farms (holding at least 4–20 ha of land) in delocalized geographies of Gauteng province, South Africa were randomly selected and assessed for the impact of FAW during the 2017 outbreak. A biotype of S. frugiperda that significantly destroyed Bt maize and non-Bt maize and exhibited tolerance to pesticides under field conditions is reported. It is observed that, whether maize at the tasseling stage carried the Cry1Ab trait or not, S. frugiperda caused a minimum damage of ~62.6% (P < 0.05, P = 0.0001, |t| = 2.78) and maximum damage of ~83.33% (P < 0.05, P = 0.0001, |t| = 2.78) in farms without refuge strategies. Phylogenetic analysis using the mitochondrial cytochrome oxidase I gene, revealed divergent evolution amongst reported S. frugiperda lineages, the emergence of singleton, and distinct haplotypes within the African continent. It is concluded that S. frugiperda that invaded maize farms in the Gauteng region of South Africa is genetically evolved from those identified in Sao Tome and Principe, and Nigeria, and tolerant to certain insecticides. Thus, the African continent might have been invaded by several routes and distinct haplotypes.