Objective: We assessed DNA conservation using a range of archaeological skeletal samples from Sudan (Missiminia in Upper Nubia, 350 B.C.E to 1400 C.E) from the unfavorable conditions of the Saharan milieu and humidity of the Nile valley by tracking maternal lineage on the 'X-Group' (Ballaneans).Method: We were able to extract, amplify, and sequence mt-DNA HVS-I (Sanger sequencing method) from 11 petrous bone samples, eight for the X-Group set and three for the reference set (one Christian, one Late Meroitic, and one Meroitic).Results: It was possible to find the haplogroups (L1b, L2, L3, H2, N, T1a, X and W) and to carry out comparative data analysis in relation to haplogroup data cited in the literature. This investigation into the maternal lineage of X-Group (350 to 500 C.E.) origins allowed us to validate the efficiency of petrous bone sampling from ancient human remains from the Nile-Saharan milieu and established that the Ballaneans experienced an in-situ development with more admixture from the Levant region and North Africa.Conclusions: Our study used mt-DNA (HVS-I) to look for the biological origins of the X-Group from Upper-Nubia and demonstrated the feasibility of ancient DNA research on skeletons from the Nile-Saharan environment. The use of Next Sequencing Generation (NGS) should optimize and improve the detection of shorter DNA strands and their sequencing in complete genomes from ancient skeletal remains (petrous bones) from hot and humid environments.