Superior inbred clones selected in S1 families can integrate an individual reciprocal recurrent selection program in sugarcane by eliminating the genetic load of the population and exploring superior hybrid combinations. Molecular markers can be used for reliable identification of the true selfing-derived clones in these S1 populations. The objective of this study was to confirm true self-fertilized individuals in sugarcane families using microsatellite markers aimed at the use of self-fertilized plants in an individual reciprocal recurrent selection strategy. Self-fertilized individuals from five cultivars were genotyped with eight simple sequence repeat (SSR) markers. The markers generated 62 polymorphic markers, with an average of seven polymorphic alleles across the cultivars tested. Three loci revealed highly informative bands and were used to assess the level of selfing in five S1 families. Selfing in these families ranged from 71.7 to 97.6%. The SSR loci provide a reliable and accurate method to identify S1 progenies in sugarcane crosses and can be used as a tool to assist selection strategies in sugarcane breeding programs.