This study is aimed to estimate and compare the inter-and within-breed variability of duck populations under genetic conservation programmes. The following four duck breeds were analysed: Germanata Veneta (AGV) and Mignon (AMG) from Italy, Pekin Krajowy (33P) and Pomniejszona (2K) from Poland. The characterisation of the four populations was carried out through a panel of 23 microsatellite markers. The analysis involved 180 individuals: 39 for AGV, 41 for AMG, 50 for 33P and 50 for 2K. An average of 11.36 alleles per locus was identified. Twenty-two loci showed high values of polymorphism information content from 0.575 to 0.912, while CAUD136 was monomorphic for the Italian breeds. The breeds showed relatively high heterozygosity: higher for the Polish populations (0.6920 for 33P and 0.6521 for 2K), and lower for the Italian (0.4497 and 0.3718 for AGV and AMG, respectively). The inbreeding coefficient was higher for the Italian breeds, AMG in particular (0.133, 0.097 and 0.121), as well as the differentiation index (0.253). The Nei's minimum distances (D M) and Reynolds distances (D R) were low between the Polish populations (0.131 and 0.088, respectively); these were associated to AGV (D M ¼ 0.191 and D R ¼ 0.259 for 33P; D M ¼ 0.174 and D R ¼ 0.226 for 2K). Finally, AGV was distant from AMG (D M ¼ 0.259 and D R ¼ 0.317). The molecular coancestry, or mean kinship was higher for the Italian breeds compared to Polish populations. The Italian populations showed intermediate values. The obtained results can be perceived as an important tool for the applied genetic conservation programmes.