The aim of this study was to analyze the variation of different genetic diversity measures in 5 Italian local chicken breeds over a 4-yr period of conservation. The local breeds were Ermellinata di Rovigo, Padovana, Pépoi, Robusta Lionata, and Robusta Maculata. A total of 368 samples were chosen for the analysis among the birds hatched in the years 2002 and 2006. Genetic variation over 30 microsatellite loci was analyzed. All 30 microsatellites were polymorphic, with a total number of alleles equal to 251, a mean (±SD) of 8.367 ± 3.378 across populations and 3.233 ± 1.338 within population. After 4 yr of conservation, a loss of alleles occurred for all the microsatellites, with the exception of 4 loci. The total number of alleles and expected heterozygosity estimates significantly decreased during the 4 yr of conservation, whereas no significant differences were detected for the microsatellites polymorphism information content or for the observed heterozygosity estimates. A decrease of the inbreeding coefficient occurred for all the breeds, with the exception of Padovana and Robusta Lionata. All populations showed evidence of a persistent significant deviation from Hardy-Weinberg equilibrium caused by an excess of homozygotes, except Robusta Maculata, which reached the equilibrium in 2006. For Pépoi, Padovana, Ermellinata di Rovigo, and Robusta Maculata, molecular coancestry increased significantly (P < 0.001) after the 4 yr of conservation. No evident genetic structures were detectable within breed, both for 2002 and for 2006 individuals. However, a slight increase in the proportion of membership for each breed had occurred in the year 2006 compared with the data obtained in the year 2002. As consequence, within breed, individuals in 2006 appear more homogeneous, producing clearer, more distinctive and separated groups. Molecular markers analysis helped us monitor the genetic variability of local breeds involved in a conservation scheme, enabling the planning of new strategies for the improvement of in situ conservation schemes.