During more than 50 years the vegetative propagation has been the form of maintaining and multiplication of the cv. 'Italy' vineyards, a 'Bicane' x 'Muscat Hamburg' hybrid. In the current study, polymorphism in 17 microsatellite loci was used to evaluate the genetic stability at DNA level in vineyards of cv. 'Italy' planted in different regions of the states of Parana and Sao Paulo, Brazil. Unchanged and equal allele frequency indicating genetic stability was reported in 47% of the microsatellite loci in vineyards of six localities, while allele frequency variation has been observed in Scu15vv, Udv44, Udv74, Udv96, Udv107, Udv108, Vvmd5, Vvmd6 and Vvs3 microsatellite loci. Alleles Udv96(140) and Vvs3(448), detected in vines in only one of the vineyards, evidenced somatic mutations at molecular level in cv. 'Italy'. Genetic diversity, as result of changes in the allele frequencies in 53% of microsatellite loci, was detected more frequently than somatic mutations due to new alleles. Polymorphism in microsatellite loci revealed different genetic stability in vineyards of cv. 'Italy' cultivated in six different Brazilian regions and indicated vineyards with less genetic stability as a possible source of somatic mutants, showing traits of agronomic interest with a potential to generate new cultivars.