As the second species used for commercial coffee, evaluation of Coffea canephora Pierre ex Froehner population genetics is a challenging task for coffee breeding. This study examined the pattern of genetic variability and genetic relationships of cultivated and wild populations of C. canephora sampled across the Guineo-Congolese region of Africa and some improved populations maintained in field genebanks. A total of 293 individuals, sampled from 17 populations, were genotyped with 39 nuclear microsatellite markers. Genetic diversity and structure were investigated with both a model-based and a graphical approach; isolation by distance was also tested. Relationships between the diversity clusters are discussed with regard to differentiation due to several glacial refuges during the Last Glacial Maximum (LGM). High genetic diversity within C. canephora is confirmed with a mean number of alleles of 11.85 per marker, a mean gene diversity of 0.72 and a mean observed heterozygosity of 0.36. An overall structure of two main groups (Guinean and Congolese) subdivided in six subgroups (2 for the Guinean and 4 for the Congolese) was found, including one described for the first time in the Guinean group. A fine structure within the Guinean group was also newly detected. Genetic structure of C. canephora appears to be consistent with its geographic repartition at the continent scale. Structure of diversity was found in accordance with localizations of refuge zones during LGM and migration from this period. Results from this genetic structure study raise our capabilities to better manage and use the collections of genetics resources for breeding purposes. Those results will be used in future association studies to optimize the number of genotypes to be phenotyped.