The success of sugarcane (Saccharum spp.) breeding programs depends on the choice of productive parent lines that have a high industrial yield and are genetically divergent. This study assessed the genetic divergence and population structure of sugarcane accessions that are the parents of the RB05 Series of the Sugarcane Breeding Program of Brazil. The DNA of 82 accessions was evaluated using 36 simple sequence repeat markers. The Jaccard similarity coefficient and Unweighted Pair Group Method with Arithmetic Mean clustering method were used to generate a cluster that was divided into 17 distinct groups derived from probabilistic models. The similarity coefficient used in both cases showed that the degree of similarity varied from 0.4716 (RB971551 x RB965586) to 0.9526 (RB936001 x SP89-1115), with a mean of 0.8536. This result demonstrates a high similarity between the 82 accessions and confirms Wright’s F statistic (0.125), which indicates moderate genetic variability. The less-similar crosses suggest that breeders seek a higher number of crosses using cultivar RB965586, highlighting the RB971551 x RB965586 and RB965586 x RB855511 crosses. The results demonstrate that crosses such as RB936001 x SP89-1115 and RB945954 x RB896342 should be avoided because of their high genetic similarity.