The human Y-chromosome haplogroup E-M81 is predominant in northwestern Africa and almost absent elsewhere, except for the Iberian Peninsula. In the present study, we investigated the internal branches of E-M81 (E1b1b1b1a) in 29 unrelated men from mainland Portugal. All individuals sampled carried the derived allele for the marker M183 (E1b1b1b1a1), and all but two had the derived allele for the marker SM001. Eleven individuals carried the derived allele for the marker Z5009 (E1b1b1b1a1c), and 16 were assigned to the paragroup E-SM001*(xZ5009, PF6794, CTS12227, M165, M5043). Median-joining networks based on seven Y-chromosome short tandem repeats (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) showed 14 different haplotypes, revealing a clear star-like structure, with the common central haplotype distributed by the three different subclades identified (M183*, SM001*, and Z5009) encompassing the typical Maghrebin core haplotype in northern African populations. The relatively high frequency of the E-M81 lineage in mainland Portugal supports a North African Y-chromosomal contribution that may have occurred during the Islamic period in the country.