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This study investigated: (1) the mitochondrial DNA (mtDNA) genetic variation in 116 unrelated individuals who originated from the Arabian Peninsula, Iran, or were of Bedouin ethnicity and (2) the genetic structure of Kuwaiti populations and compared it to their neighboring populations. These subpopulations were tested for genetic homogeneity and shown to be heterogeneous. Restriction fragment length polymorphism (RFLP) and mtDNA sequencing analyses of HVRI were used to reconstruct the genetic structure of Kuwait. The results indicated that the combined Kuwaiti population has a high frequency of haplogroup R0 (17%), J (12%), and U (12%) similar to other Arabian populations. In addition, contemporary African gene flow was detected through the presence of sub-haplogroup L (L1 and L2) (2%) and the absence of L3 which is reflective of an earlier migration. Furthermore, the multidimensional scaling (MDS) plot showed that the Kuwaiti population clusters with neighboring populations, including Iran and Saudi Arabia indicating gene flow into Kuwait. According to this study, the Kuwaiti population may be undergoing an expansion in a relatively short period of time, and the maternal genetic structure of Kuwait resembles both Saudi Arabia and Iran.