Technical Abstract: New lines of Saccharum hybrids with an array of S. spontaneum cytoplasm backgrounds are reported. To expand the genetic base of sugarcane, we made eleven bi-parental crosses between ten S. spontaneum (S) and six commercial-type sugarcane (C) clones during the 2001 crossing season. Prior to crossing, all the maternal S. spontaneum inflorescences were emasculated by immersion in a 50oC circulating water bath for 5 minutes. Analysis of microsatellite fingerprints between parents and progeny allowed us to classify 1,952 progeny grown out from these crosses into four genotypic classes. Class H progeny inherited microsatellite alleles from both the S. spontaneum and the commercial-type parents and were, therefore, considered being F1 hybrids. Class S and Class C progeny inherited microsatellite alleles only from one parent and were considered to be either selfs of either parent or F1 hybrids that only inherited allele(s) from one parent. Class X progeny inherited non-parental microsatellite allele(s) in addition to the allele(s) from the maternal S. spontaneum parent and were considered to be contaminants. With the exception of one cross, eight to ten Class H progeny were pre-selected from each cross while still in seedling greenhouse and were backcrossed with commercial-type sugarcane clones. The remaining progeny were transplanted into a breeding nursery for phenotypic evaluation that concurred with the molecular classification. Pearson Correlation Coefficients between molecular and phenotypic classifications were inconsistent that justified the need of molecular markers in the selection process. This study demonstrated that the molecular approach of fingerprinting progeny to confirm parentage prior to field planting even with only one microsatellite marker might substantially increase selection efficiency.