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The genetic diversity of the Mediterranean swordfish (Xiphias gladius Linneus) has not been explored extensively at its easternmost range so far. In this study, modern X. gladius samples from the eastern part of the Mediterranean basin, north of the Aegean Sea (Aegean‐2013, n = 26) and the Mediterranean coast of Turkey (N.Levantine‐2013, n = 42) were studied genetically, along with ancient samples from Yenikapı excavation (n = 6). Partial mitochondrial DNA control region sequences (entire sequences, clade I and clade II) were evaluated spatially and temporally together with previously published sequences (Alvarado Bremer et al., Molecular Phylogenetics and Evolution, 2005, 36, 169–187; Viñas et al., ICES Journal of Marine Science, 2010, 67, 1222–1229; Righi et al., Diversity, 2020, 12, 170) from the rest of the Mediterranean Sea. Pair‐wise FST and pair‐wise AMOVA tests showed that, in general, groups of eastern populations and western Mediterranean populations have not genetically differed from each other significantly nearly in the past 20 years. Therefore, the results direct reconsideration of previous descriptions of population sub‐structure within the Mediterranean and support high gene flow throughout the region. On the contrary, the results of this study confirmed the existence of genetic diversity differences between western and eastern Mediterranean, with eastern being low. One‐tailed permutation tests revealed that θ, which is directly proportional to long‐term female effective population size (Ne), decreased significantly (P < 0.05) in both regions over the past two decades. On the Turkish coasts, θ is not significantly different from that of the nearly contemporary eastern Mediterranean population. Nonetheless, θ of the ancient sample was consistently and significantly (P < 0.001) higher than those of the eastern and western Mediterranean populations in clade I and clade II. Furthermore, it contains two mitochondrial haplotypes that are not observed in modern samples, suggesting that the Ne of X. gladius in the eastern was high in Byzantium times. Eight microsatellite loci were also genotyped in modern samples. The microsatellite‐based present Ne estimate of the pooled Aegean‐2013 and N.Levantine‐2013 populations was lower than 1000 according to the upper limit of 95% c.i. and possibly even lower than 100 according to the mean of posterior distribution of the present Ne estimate calculated by the software package MSVAR. These alarming genetic signals for the sustainability of X. gladius on the coasts of Turkey are in agreement with the nearly collapsing X. gladius fisheries as depicted also in the fisheries statistics. Overall, congruent with the previous studies, the data presented here show that sustainability of the X. gladius population in Mediterranean is under major threat. Therefore, X. gladius around the Turkish coasts need an immediate stringent action and management plan. [ABSTRACT FROM AUTHOR]