In this study we analyzed 295 unrelated Berber-speaking men from northern, central, and southern Morocco to characterize frequency of the E1b1b1b-M81 haplogroup and to refine the phylogeny of its subclades: E1b1b1b1-M107, E1b1b1b2-M183, and E1b1b1b2a-M165. For this purpose, we typed four biallelic polymorphisms: M81, M107, M183, and M165. A large majority of the Berber-speaking male lineages belonged to the Y-chromosomal E1b1b1b-M81 haplogroup. The frequency ranged from 79.1% to 98.5% in all localities sampled. E1b1b1b2-M183 was the most dominant subclade in our samples, ranging from 65.1% to 83.1%. In contrast, the E1b1b1b1-M107 and E1b1b1b2a-M165 subclades were not found in our samples. Our results suggest a predominance of the E1b1b1b-M81 haplogroup among Moroccan Berber-speaking males with a decreasing gradient from south to north. The most prevalent subclade in this haplogroup was E1b1b1b2-M183, for which differences among these three groups were statistically significant between central and southern groups.