Recent studies on Atlantic swordfish (Xiphias gladius L. 1758) genetic structure have demonstrated significant heterogeneity but the precise boundary between populations remains to be identified. In this context, genetic diversity was investigated by PCR-RFLP analysis at the control region of mitochondrial DNA (D-loop) from 274 swordfish specimens collected from five different areas of the Atlantic Ocean. The analysis of molecular variance (AMOVA) showed that genetic variation was mainly due to differences within rather than between the studied areas. Additionally, the phylogenetic analysis did not show evident relationships among haplotypes from all areas. However, low but significant FST values were recorded when comparing Equatorial samples with those from the north central and north tropical Atlantic. These results do not support a need for changing the current management boundary for the Atlantic fishery.