A detailed dissection of R-DF27 paternal lineage is performed for the first time. DF27 haplogroup seems to have a geographical significance in the Iberian Peninsula. The TMRCAs suggest DF27 is a young lineage that arose 4176 ± 696 years ago. DF27 could be used to trace Iberian male migrations into the Americas. DF27 could be used to trace the biogeographic paternal origin of a forensic evidence.