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Simple Summary: The Arabian horse is probably one of the most historic horse breeds worldwide. It has been selectively bred by the Bedouin tribes in the steppes of Arabia for over 2000 years. Bedouin tribes have created their own classification of strains based on maternal lines. The aim of our study was to investigate genetic differentiation at the level of (i) the current populations (Desert-Bred, Straight Egyptian and Polish Arabians) and (ii) the traditional Bedouin strains. Two hundred and eleven Arabian individuals, representing 12 strains, were sampled. Mitochondrial DNA D-loop analysis showed higher genetic diversity in the Egyptian and Polish Arabian populations than in the Desert-Bred. No genetic markers distinguishing the traditional strains were highlighted in this study, a fact probably due to the lack of interest in this component in modern breeding programs. Some Polish Arabian individuals, who could not be traced back to the Bedouin tribes, appeared to be genetically distinct from the other studied horses, requiring more in-depth study. This research, complemented by future SNP and Y-chromosome analyses, may provide a more accurate evaluation of the relationships between Arabian horse populations′ genetic and traditional classification. Genetic diversity and phylogenetic relationships within the Arabian show horse populations are of particular interest to breeders worldwide. Using the complete mitochondrial DNA D-loop sequence (916 pb), this study aimed (i) to understand the genetic relationship between three populations, the Desert-Bred (DB), a subset of the Kingdom of Saudi Arabia (KSA), United Arab Emirates (UAE) and Bahrain (BAH), the Straight Egyptian (EG) and the Polish bloodline (PL), and (ii) to assess the accuracy of the traditional strain classification system based on maternal lines, as stated by the Bedouin culture. To that end, we collected 211 hair samples from stud farms renowned for breeding Arabian show horses from Nejd KSA, Bahrain, Egypt, Qatar, Morocco, UAE, and Poland. The phylogenetic and network analyses of the whole mitochondrial DNA D-loop sequence highlighted a great genetic diversity among the Arabian horse populations, in which about 75% of variance was assigned to populations and 25% to strains. The discriminant analysis of principal components illustrated a relative distinction between those populations. A clear subdivision between traditional strains was found in PL, in contrast to the situation of DB and EG populations. However, several Polish horse individuals could not be traced back to the Bedouin tribes by historical documentation and were shown to differ genetically from other studied Bedouin strains, hence motivating extended investigations. [ABSTRACT FROM AUTHOR]