Thorough characterization of the genetic variability in bread wheat (Triticum aestivum L.) is important for a better improvement of this key crop and to increase cereal yield in the context of sustainable agriculture to face human needs in the next decades. To study the genetic variability of SSRs on wheat homoeologous group 3 chromosomes, we characterized 38 hexaploid and two tetraploid wheat lines using a set of 165 microsatellites that we cytogenetically assigned to the 17 deletion bins for chromosomes group 3. A comparative analysis of the genetic variability through the PIC value study, allele numbers and SSR lengths indicated that there were no statistically significant differences (p > 0.05) between the three chromosomes of this homoeologous group despite the fact that SSRs from chromosome 3B exhibited slightly more alleles per locus compared to chromosomes 3A and 3D as well as slightly higher PIC values compared to chromosome 3D. However, there was a stronger correlation between SSR length and allele number on the short arms compared to the long aims and the correlation increased from the centromeres toward the telomeres. We did not find statistically significant differences in allele numbers and PIC values for SSRs located in more distal bins on 3A and 3B chromosomes. On the contrary, for chromosome 3D, we observed significant differences (p < 0.05) between the PIC values determined for SSRs assigned to deletion bin 3DL3-0.81-1.00 bin that is located distal compare to the more proximal region (C-3DL3-0.81). These results suggest that recombination which is higher in the telomeric regions does not contribute to increase a lot the variability of the SSRs.