Biomphalaria pfeifferi is a key intermediate host for Schistosoma mansoni transmission in Sudan. In total, 27 complete mitochondrial genomes from seven B. pfeifferi populations in Gezira State, Sudan, were sequenced for the first time to investigate their population structure and phylogenetic relationships. This involved comparing the nucleotide composition, codon usage, rRNAs, and tRNAs of the East Gezira (EG), South Gezira (SG), Hasahisa (HA), Greater Wad Medani (GW), Managil (MA), and North Umelgura (NU1, NU3) populations. All 27 mitogenomes (13,688–13,696 bp) contained 37 genes with conserved AT/GC content (76.7/23.4%). Phylogenetic analysis revealed that although samples clustered within the same clade, B. pfeifferi from EG, SG, NU1, and NU3 grouped closely with B. pfeifferi from Kenya, whereas HA and GW samples formed distinct ancestral lineages. The MA population exhibited unique genetic characteristics, supported by phylogenetic trees and nucleotide/amino acid identity, suggesting the potential presence of a distinct B. pfeifferi subspecies that warrants further investigation. All protein-coding genes evolved under negative selection, with the amino acids of nad1 and nad6 being highly conserved, while nad3 exhibited some variation. Further research on the mitogenomic diversity of B. pfeifferi and other Biomphalaria species in Sudan and across Africa is needed in order to better understand the population structure and evolutionary history of Biomphalaria.