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There are several factors, such as genetic drift, gene flow and migration that affect the population genetic structure and phylogeographic distribution of genetic lineages within single species. Previous studies of the bladder grasshoppers, Bullacris unicolor of South Africa, showed divergence in mitochondrial CO1 (cytochrome c oxidase subunit 1) haplotype diversity and significant genetic structure. In this study, we revisit these findings adding more samples from different locations and using mitochondrial CO1 and Internal transcribed spacer (ITS) gene sequences. We tested the hypothesis that the western, northern and eastern distribution ranges of B. unicolor show different population genetic patterns, corresponding with isolation-by-distance. Mitochondrial CO1 and ITS data were collected for 99 individuals from 12 localities across the Western, Northern and Eastern sides of South Africa. Overall, significant variation in genetic structure was found across the localities as indicated by F
ST
analyses. Haplotype and phylogeographical analyses suggested that restricted gene flow played a role in shaping current genetic patterns, although isolation-by-distance was not supported, as confirmed by Mantel tests. Phylogenetic trees of both genetic sequences revealed two major clades, with western and northern sides. Also, the major clades exhibit a few sub-clades within the localities, showing other factors shaping the genetic structure of B. unicolor include the geographical barriers, and most likely due to changes in habitat specificity and habitat fragmentation. Taken together, this study aims to contribute information on the population structure and genetic diversity of B. unicolor populations across South Africa.