Haplogroup E3b is defined by a single nucleotide mutation (SNP) in locus M35 and is found at high frequency (more than 35%) in populations from North Africa with a heterogeneous distribution. On the basis of compilation of 553 Y-chromosomes from Europe and 633 from sub-Saharan Africa we selected 130 individuals belonging to haplogroup E3b and characterized subhaplogroups according to the Y-Chromosome Consortium nomenclature. Y-chromosome haplotypes can be defined using short tandem repeats (STR). The use of STRs makes it possible to measure diversity and estimate age coalescence. Significant differences on frequencies of Y-chromosome STR loci were found among the E3b subhaplogroups and the same was observed when haplotype frequencies were considered. Some mutations in SNPs were detected when comparing E3b subhaplogroups with the correspondent STR haplotypes. These results show that the mutation rate for some SNPs could be higher than previously thought and also that it is important to associate both haplotype and haplogroup in Y-chromosome studies.