The genetic base of today's sugarcane cultivars appears to be narrow and could be the reason for current slow progress in improving sugarcane crops. Sixty-three primer pairs (producing 263 polymorphic fragments) flanking simple sequence repeats or micro-satellites were used for assessing the genetic variability of five S. officinarum clones and 33 sugarcane cultivars used in CENICANA breeding projects, selected for their economic and agronomic importance in several Central and South-American countries. NTSYS (Numerical Taxonomy and Multivariate Analysis System) and SAS (Statistical Analysis System) statistical software was used to analyse data. The number of alleles recorded per marker ranged from 1 to 16 (mean = 5). Cluster analysis showed a clear separation of cultivars from S. officinarum clones. The average of genetic similarity between sugarcane genotypes studied was 0.664, while genetic diversity analysis revealed a very different group (H: 0.973). An interesting results concerned CC 91-1880 distribution very close to that of Q genotypes from Australia and also S. officinarum clones, suggesting that this cultivar would be a good candidate for further studies by breeders. The results obtained are useful for CENICANA's breeding program because, in spite of the genetic homogeneity present in today's sugarcane cultivars, it is clear that allelic variants are present in some of these cultivars and could be used in the new breeding projects.