Bemisia tabaci (Gennadius) is a polyphagous, highly destructive pest that is capable of vectoring viruses in most agricultural crops. Currently, information regarding the distribution and genetic diversity of B. tabaci in South Sudan is not available. The objectives of this study were to investigate the genetic variability of B. tabaci infesting sweet potato and cassava in South Sudan. Field surveys were conducted between August 2017 and July and August 2018 in 10 locations in Juba County, Central Equatoria State, South Sudan. The sequences of mitochondrial DNA cytochrome oxidase I (mtCOI) were used to determine the phylogenetic relationships between sampled B. tabaci. Six distinct genetic groups of B. tabaci were identified, including three non-cassava haplotypes (Mediterranean (MED), Indian Ocean (IO), and Uganda) and three cassava haplotypes (Sub-Saharan Africa 1 sub-group 1 (SSA1-SG1), SSA1-SG3, and SSA2). MED predominated on sweet potato and SSA2 on cassava in all of the sampled locations. The Uganda haplotype was also widespread, occurring in five of the sampled locations. This study provides important information on the diversity of B. tabaci species in South Sudan. A comprehensive assessment of the genetic diversity, geographical distribution, population dynamics, and host range of B. tabaci species in South Sudan is vital for its effective management.